Surface antigens and proteins useful in compositions for the diagnosis and prevention of Lyme disease

ABSTRACT

A novel isolated surface antigen, which is expressed in vitro by spirochetes of  Borrelia garinii  strain IP90, is characterized by a relative molecular mass of 39.5 kDa and the ability to induce antibodies which kill spirochetes of the  B. burgdorferi  sensu lato strain IP90 by ADCK in vitro. Novel  B. garinii  cassette string protein or fragments thereof are also useful, as is the P39.5 protein in diagnosing Lyme disease and in compositions for treatment or prophylaxis thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/445,803, filed Dec. 13, 1999, which is a 371 ofInternational Patent Application No. PCT/US98/13551, filed Jun. 29,1998, which claims the benefit of the priority of U.S. ProvisionalPatent Application No. 60/051,271, filed Jun. 30, 1997, now abandoned.

[0002] This invention was funded in part by the National Institutes ofHealth Grant Nos. ROI AI35027 and RR/AE00164-32. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The bacterium Borrelia burgdorferi (sensu lato) is the causativeagent of Lyme borreliosis, i.e., Lyme disease. This disease istransmitted by the bite of various species of Ixodes ticks carrying thespirochete. The main reservoir of the infection in the United States isthe white footed mouse, Peromyscus leucopus, and the infection can betransmitted to many mammalian species including dogs, cats, and man [J.G. Donahue, et al, Am. J. Trop. Med. Hyg., 36:92-96 (1987); R. T. Green,et al, J. Clin. Micro., 26:648-653 (1988)]. Despite the presence of anactive immune response, the disease persists for years in patients. Suchpersistence is postulated to be the result, at least in part, ofantigenic variation in the bacterial proteins [J. R. Zhang et al, Cell,89:275-285 (1997)].

[0004] The diagnosis of Lyme disease in humans and animals has beencompromised by the lack of definitive serology leading to rapid andaccurate testing. Current diagnostic tests suffer from low sensitivityand specificity, as illustrated by a recent survey of diagnosticlaboratories' performance issued by the Wisconsin State Laboratory ofHygiene [L. Bakken et al, J. Clin. Microbiol., 35:537 (1997)]. A simple,sensitive and specific diagnostic composition and method for earlydetection of Lyme disease is needed in the art.

[0005] Publications relating to proteins and polypeptides of Borreliaburgdorferi have suggested their use as diagnostic or pharmaceuticalagents. Such proteins and polypeptides include outer surface proteins Aand B (OspA and OspB), flagellin, and other proteins designated P21,P39, P66, and P83 according to their estimated molecular weights [A. G.Barbour et al, Infect. Immun., 45:94-100 (1984); W. J. Simpson et al, J.Clin. Microbiol., 28:1329-1337 (1990); K. Hansen et al, Infect. Immun.56:2047-2053 (1988); K. Hansen et al, Infect. J. Clin. Microbiol.,26:338-346 (1988); B. Wilske et al, Zentral, Bakteriol, Parasitenkd,Infektionshkr. Hyg. Abt. 1 Orig. Reihe. A., 263:92-102 (1986); D. W.Dorward et al, J. Clin. Microbiol., 29:1162-1170 (1991); published NTISUS patent application No. 485,551; European patent application No.465,204, published Jan. 8, 1992; International Patent Application No.PCT/US91/01500, published Sep. 19, 1991; International PatentApplication No. PCT/EP90/02282, published Jul. 11, 1991; InternationalPatent Application No. PCT/DK89/00248, published May 3, 1990;International patent Publication No. WO92/00055, published Jan. 9,1992].

[0006] A preferred protein candidate for a vaccine is OspA [M. Philippet al, J. Spirochetal and Tick-borne Diseases, 3:67-79 (1996)]. Theexpression of OspA is either abrogated or down-regulated when thespirochetes are en route from the tick's midgut to the salivary glands,as blood feeding is taking place [A. DeSilva et al, J. Exp. Med.,183:271-275 (1996)]. This phenomenon generates potential problems thatmay diminish the OspA vaccine's efficacy. Spirochetal attrition mayoccur only within the tick midgut [A. DeSilva et al, cited above] andnot upon infection of the vertebrate host. Because the saliva of Ixodesscapularis contains a decomplementing factor [T. Mather et al, “Ixodessaliva: vector competence for Borrelia burgdorferi and potential vaccinestrategies”, in VII International Congress on Lyme Borreliosis, SanFrancisco, Calif. (1996)], spirochetal attrition within the tick'smidgut might occur via a mechanism involving only antibody and notcomplement.

[0007] Although the mode of action of antibody-dependent killing is notfully understood, it appears to be a less efficient mechanism of killingthan that mediated by antibody and complement, acting together [M. Soleet al, Infect. Immunol., 66:2540-2546 (1998)]. Thus, it may permitevasion from the midgut of those spirochetes that have a low surfacedensity of OspA. Indeed, although OspA is an abundant B. burgdorferiprotein, only a minor fraction of OspA molecules is exposed on the outersurface of the spirochete [D. Cox et al, Proc. Natl. Acad. Sci.,93:7973-7978 (1996)]. Thus, small variations in the absolute number ofOspA surface molecules may cause significant differences in thespirochete attrition rate and make it possible for a fraction of theresident spirochetes to abscond to the salivary glands.

[0008] OspA escape mutants will readily avoid killing altogether, and ifthey are infectious to the vertebrate host, they will contribute todiminish the OspA vaccine efficacy even further. In clonal populationsof B. burgdorferi which are allowed to grow in vitro, the prevalence ofmutants that resist killing by anti-OspA antibody ranges between 10⁻⁵and 10⁻² [A. Sadziene et al, J. Exp. Med, 176:799-809 (1992)]. If suchfrequencies are reproduced in a feeding nymph, in which spirochetenumbers may reach a mean of 7,848 within 15 hours of attachment [A.DeSilva et al, Am. J. Trop. Med. Hyg., 53:397-404 (1995)] then severalmutants may be present in a single tick. Typical mutant phenotypesinclude those that express neither OspA nor OspB and, frequently,expressors of a chimeric molecule composed of an N-terminal fragment ofOspA fused to a C-terminal fragment of OspB. These deletion mutants havebeen found in multiple strains of B. burgdorferi [P. Rosa et al, Mol.Microbiol., 6:3031-3040 (1992)] and in several tick isolates fromCalifornia [T. Schwan et al, J. Clin. Microbiol. 31:3096-3108 (1993)].Chimeric (deletion) mutants, which are able to resist killing withanti-OspA antibody alone, are killed by the combined action of antibodyand complement. Since complement appears to be nonfunctional within thetick [T. Mather et al, cited above], and OspA, and probably its chimericform as well, are not expressed within the vertebrate host shortly afterinfection, chimeric OspA escape mutants could infect the vertebratehost. Further, it is estimated that up to 2% of Ixodid ticks are onlypartially fed and are therefore still questing [Y. Lobet, personalcommunication]. If such ticks have taken their incomplete meal from a B.burgdorferi-infected host, they will have spirochetes in their salivaryglands which do not express OspA and which will readily infect anOspA-vaccinated host.

[0009] No booster effect has been observed [M. Philipp et al, citedabove] nor should one be expected upon spirochetal challenge of anOspA-vaccinated host. Hence, the OspA vaccine may require repeatedadministrations to maintain effective antibody titers.

[0010] There is a thus a need in the art for additional, and improved,methods and compositions for prevention of Lyme disease in humans andanimals, and for treatment thereof

SUMMARY OF THE INVENTION

[0011] The present invention satisfies the need in the art by providingmethods and compositions for prevention of Lyme Disease based onspirochetal antigens that are expressed by the spirochete during itsresidence in the vertebrate host. Such methods and compositions may alsobe employed for the treatment of Lyme Disease.

[0012] In one aspect, the invention provides an isolated B. gariniisurface antigen which is expressed in vivo by the spirochete in thevertebrate host. The antigen, referred to herein as P39.5, is furthercharacterized as having a relative molecular mass of 39.5 kDa. Fragmentsof this antigen are also useful in the compositions and methods of thisinvention.

[0013] In another aspect, the invention provides novel B. gariniicassette string polypeptides/proteins.

[0014] In yet another aspect, the invention provides nucleic acidsequences encoding P39.5 or a fragment thereof, such as P7-1, as well asnucleic acid sequences encoding other B. garinii cassette stringproteins or fragments thereof. These nucleic acid sequences includesequences which hybridizes to the above sequences under stringentconditions, allelic variants thereof and deletion mutants thereof.

[0015] In another aspect, the invention provides novel proteinscomprising fragments of the P39.5 protein sequence or cassette stringprotein sequences described above, optionally fused to, or mixed with, asecond selected polypeptide or protein, which may be a protein of up toabout 90% identity in its amino acid sequence with that of P39.5, orother Borrelia antigens, such as OspA, and proteins or polypeptidesderived from other microorganisms.

[0016] In still another aspect, the invention provides novel proteincompositions comprising the protein sequences of the antigen(s)described above, or fragments thereof, optionally mixed with a secondselected polypeptide or protein, which may be a protein of up to about90% identity in its amino acid sequence with that of P39.5, or otherBorrelia antigens, such as OspA, and proteins or polypeptides derivedfrom other microorganisms.

[0017] In still a further aspect, the invention provides a method forrecombinantly producing the above-described P39.5 protein, fragmentsthereof or fusion proteins containing such fragments, by expressing aDNA sequence encoding the protein, fragment or fusion protein in aselected host cell, and isolating the protein therefrom. Host cellstransformed with such DNA sequences are also provided herein.

[0018] In still another aspect, the invention provides an isolatedantibody directed against the above-described P39.5 antigen, fragmentsthereof, cassette string proteins or fragments, or a fusion proteincontaining such fragments. These antibodies may be polyclonal. Thepresent invention also provides a method for producing such antibodiescomprising immunizing a human or an animal with an isolated antigen asdescribed above, or with one or a mixture of more than one fragment(s)thereof, or fusion protein containing one or more fragment(s) of thisinvention. Other types of antibodies may be prepared from such immunizedanimals, e.g., recombinant, monoclonal, chimeric, humanized, etc.

[0019] In another aspect, the invention provides a therapeuticcomposition and methods for treating humans and/or animals with Lymedisease. The therapeutic composition contains an antibody, or protein,or fragment as described above and a suitable pharmaceutical carrier.

[0020] In a further aspect, the invention provides vaccine compositionsand methods of vaccinating a human or animal patient against LymeDisease by use of these above-described compositions. The compositionscontain an effective amount of at least one B. garinii antigen of thisinvention, e.g., an antigen that is expressed in vitro by Borreliagarinii strain IP90 spirochetes, said antigen having a relativemolecular mass of 39,500 daltons, or antigenic fragment(s) thereof, or afusion protein containing such a fragment, or cassette stringprotein(s), and a pharmaceutically acceptable carrier. The vaccinecomposition may contain the P39.5 protein, fragments thereof, fusionproteins or mixtures of proteins as described above.

[0021] In yet a further aspect, the invention provides vaccinecompositions and methods of vaccinating a human or animal patientagainst Lyme Disease by use of nucleic acid compositions, e.g., DNAvaccines. The compositions contain an effective amount of a DNA sequenceencoding at least one B. garinii antigen of this invention or antigenicfragment(s) thereof, cassette string antigen(s), or a fusion proteincontaining such a fragment, and an optional pharmaceutically acceptablecarrier.

[0022] In yet a further aspect, the invention provides a method fordiagnosing Lyme borreliosis in a human or animal. This method includesthe steps of incubating an antigen or antibody of this invention,preferably conventionally labeled for detection, with a sample ofbiological fluids from a human or an animal to be diagnosed. In thepresence of B. burgdorferi infection of the human or animal patient, anantigen-antibody complex is formed. Subsequently the reaction mixture isanalyzed to determine the presence or absence of these antigen-antibodycomplexes. In a further embodiment, the diagnostic assay employs DNAsequences, preferably anti-sense sequences, of the antigen or fragmentsthereof, and diagnoses infection by the presence of sequences in abiological fluid from the patient that hybridizes thereto. Otherconventional assay formats may be employed using reagents identified bythis invention.

[0023] In another aspect the invention provides a kit for diagnosinginfection with B. burgdorferi in a human or an animal patient samplewhich contains at least one antibody capable of binding at least oneantigen of this invention of antigenic fragment(s) thereof, or a DNAsequence encoding one or more antigen(s) of this invention or ananti-sense sequence thereof. The antibodies and sequences may beoptionally labeled for detection, or a detection system may be includedin the kit.

[0024] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a graph of the antibody dependent, complement-mediatedkilling (ADCK) titration of spirochetes from B. burgdorferi strains JD1(∘), B31 (Δ), and B. garinii strain IP90 (□) with serum from a rhesusmonkey tick-inoculated with JD1 spirochetes.

[0026]FIG. 2 is a schematic depiction of portions of the DNA sequence ofthe novel P39.5 encoding a single open reading frame which encodes adeduced protein of 37.7 kDa. This DNA fragment is named 7-1, and thededuced protein referred to as P7-1. The region in black is a uniqueregion spanning about bp769 to about bp854 of SEQ ID NO: 1. The regionlabeled IA spans about bp 1 to about bp 309 of SEQ ID NO: 1; IB spansabout bp 855 to about bp 1189 of SEQ ID NO: 1). Regions IA and IB have a70% identity. Regions IIA, which spans about bp 310 to about bp494 ofSEQ ID NO: 1 and IIB, which spans about bp 595 to about bp 769 of SEQ IDNO: 1, have a 91% identity. Regions A, which spans about bp 208 to aboutbp 309 of SEQ ID NO: 1, and B, which spans about bp 495 to about bp 595of SEQ ID NO: 1, have an 84% identity. Regions B and C, which togetherspan about bp 1090 to about bp1189 of SEQ ID NO: 1, have a 90% identity.

[0027]FIG. 3 is a bar graph which illustrates in vitro killing of IP90spirochetes with: plasma of a monkey infected with JD1 spirochetes andmonkey complement (bar 1); antibody from the same plasma which isaffinity purified on native P39.5 and monkey complement (bar 2); thesame antibody as described for bar 2 without complement (bar 3);complement alone (bar 4); and BSK-H medium alone (bar 5). Error barsrepresent the standard deviation of the mean of two determinations.

[0028]FIG. 4 is a bar graph showing ADCK of spirochetes of the IP90strain using: a plasma pool from monkeys infected with B. burgdorferiJD1 at a dilution of 1:10 (bars 1, 5); pooled serum from mice immunizedwith a recombinant form of P7-1 (rP7-1) and Ribi adjuvant at 1:10 (bars2,6); and 1:50 (bar 3); and mice immunized with Ribi adjuvant alone(bars 4,7). Monkey complement was used in ADCK of bars 1-4 and guineapig complement in ADCK of bars 5-7. Error bars represent the standarderror of the mean of two determinations.

[0029]FIG. 5 is an illustration of the ‘string’ of silent VLS cassettesin a stretch of DNA about 8 kb in length, duplicated from J. R. Zhang etal, Cell, 89:275-285 (1997).

[0030]FIG. 6 is a Western blot of lysates from spirochetes of B.burgdorferi strains JD1 and B31, and B. garinii strain IP90 developedwith serum from monkeys needle-inoculated (lanes 1) and tick-inoculated(lanes 2) with the JD1 spirochetes, and antibodies from the latter serumaffinity purified off of whole live JD1 spirochetes (lanes 3).

SEQUENCE LISTING DESIGNATIONS

[0031] SEQ ID NO:1 is the nucleic acid sequence of clone 7-1.

[0032] SEQ ID NO:2 is the deduced protein sequence of clone 7-1.

[0033] SEQ ID NO:3 is the 5′ end of the nucleic acid sequence of clone1-1.

[0034] SEQ ID NO:4 is a partial nucleic acid sequence of clone 1′-1,found in the middle of the nucleic acid sequence.

[0035] SEQ ID NO:5 is the 3′ end of the nucleic acid sequence of clone1-1.

[0036] SEQ ID NO:6 is the 5′ end of the nucleic acid sequence of clone3-1.

[0037] SEQ ID NO:7 is the 3′ end of the nucleic acid sequence of clone3-1.

[0038] SEQ ID NO:8 is the 5′ end of the nucleic acid sequence of clone6-1.

[0039] SEQ ID NO:9 is the 3′ end of the nucleic acid sequence of clone6-1.

[0040] SEQ ID NO:10 is the 5′ end of the nucleic acid sequence of clone9-1.

[0041] SEQ ID NO:11 is the 5′ end of the nucleic acid sequence of clone12-1.

[0042] SEQ ID NO:12 is the 3′ end of the nucleic acid sequence of clone12-1.

[0043] SEQ ID NO: 13 is a partial nucleic acid sequence obtained fromclone 14 which, when added to the 5′ terminus of clone 7-1 [SEQ ID NO:1], forms a larger fragment of P39.5, i.e., P7-1. The first 5′ terminalsix bases are from the plasmid vector.

[0044] SEQ ID NO: 14 is the deduced protein sequence of SEQ ID NO: 13.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates generally to the field ofpharmaceutical and diagnostic compositions useful in the diagnosis,treatment and prophylaxis of Lyme borreliosis. More specifically, theinvention provides an isolated natural surface antigen of Borelliagarinii and antibodies thereto for use in the diagnosis, treatment orprevention of Lyme disease.

[0046] The present invention provides a novel Borrelia surface antigen,P39.5, that is expressed by the spirochete when it resides in thevertebrate host (“in vivo”). This antigen is targeted byantibody-dependent, complement-mediated (ADCK) killing in vitro. Thisnovel antigen, fragments thereof, antibodies developed thereto, thenucleic acid sequences encoding same, and the use of such antigens,antibodies and nucleic acid sequences in diagnostic, therapeutic andprophylactic compositions and methods for the treatment or prevention ofLyme Disease provide advantages over the use of other Borrelia proteinsand antibodies in known compositions and methods for this purpose.

[0047] I. The P39.5 Antigen of the Invention

[0048] In one embodiment, the present invention provides a novelisolated antigen, referred to as P39.5, which is expressed both in vitroand in vivo by Borrelia garinii strain IP90 spirochetes. This antigen,or a homolog thereof, is expressed in vivo by spirochetes of the B.burgdorferi sensu stricto strains JD1, B31 and NT1. This antigen has inIP90 spirochetes an apparent molecular mass of 39.5 kDa, and isfunctionally characterized by the ability to elicit antibody in animalsduring the course of a natural infection with Borrelia spirochetes,which kills IP90 spirochetes by ADCK in vitro. The elicited antibodyalso kills spirochetes of the NT₁ strain of B. burgdorferi sensu lato, astrain isolated from the cerebrospinal fluid of a patient in the UnitedStates, but not further characterized within the sensu lato complex ofspecies. This strain was provided by Dr. Patricia Coyle, StateUniversity of New York at Stony Brook, New York.

[0049] The gene fragment, designated 7-1, from Borrelia garinii strainIP90 inserted in pBluescript II plasmid was transformed in E. coli anddeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. (“ATCC”) on Jun. 27, 1997 under Accession No.98478. When this gene fragment is expressed in E. coli, isolated as apure protein and the protein used as an immunogen in mice, the antibodythus produced reacts with P39.5 of IP90. Other gene fragments,designated, 1-1,3-1, 6-1,9-1 and 12-1, from Borrelia garinii strain IP90were similarly each inserted in pBluescript II plasmids, transformed inE. coli and deposited. These latter deposits were made with the ATCC onJun. 10, 1998 under Accession Nos. 98768 for 1-1, 98769 for 3-1, 98770for 6-1, 98771 for 9-1 and 98772 for 12-1. All deposits were all made tomeet the requirements of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure, and fully comply with the requirements of the United StatesPatent and Trademark Office for deposits for patent purposes. Sequencesuseful in the present invention may be obtained from these deposits.

[0050] A. Nucleic Acid Sequences

[0051] The present invention provides mammalian nucleic acid sequencesencoding fragments of P39.5. The nucleic acid sequences of thisinvention are isolated from cellular materials with which they arenaturally associated. As described in the examples below, a segment of1190 bp which encodes a portion of the coding region of the P39.5 geneof IP90 was cloned and sequenced. It encompasses a single open readingframe that encodes a putative 37.7 kDa protein. This partial DNAsequence is reported in SEQ ID NOS: 13 and 1. SEQ ID NO: 13 is asequence immediately 5′ to the first base of SEQ ID NO: 1. Togetherthese sequences from a partial protein of 1189 bp and about 396 aminoacids. The sequenced fragment is composed of mostly hydrophilic domainsthat contain several internally repeated regions and a unique regionspanning about bp 627 to about bp 712 of SEQ ID NO: 1. See, FIG. 2,which identifies the repeated sequences and identifies the homologiesand percent identities of such sequences.

[0052] Where in this text, protein and/or DNA sequences are defined bytheir percent homologies or identities to identified sequences, thealgorithms used to calculate the percent identities or percentsimilarities include the following: the Smith-Waterman algorithm [J. F.Collins et al, 1988, Comput. Appl. Biosci., 4:67-72; J. F. Collins etal, Molecular Sequence Comparison and Alignment, (M. J. Bishop et al,eds.) In Practical Approach Series: Nucleic Acid and Protein SequenceAnalysis XVIII, IRL Press: Oxford, England, UK (1987) pp.417], and theBLAST and FASTA programs [E. G. Shpaer et al, 1996, Genomics,38:179-191]. These references are incorporated herein by reference.

[0053] Also encompassed within this invention are other nucleic acidfragments of B. garinii, e.g., cassette string fragments, as well asfragments of P39.5. Such fragments are referred to as 1-1 (which encodesa protein/peptide P1-1), 3-1 (which encodes a protein/peptide P3-1), 6-1(which encodes a protein/peptide P6-1), 9-1 (which encodes aprotein/peptide P9-1) and 12-1 (which encodes a protein/peptide P12-1).Preferably, such fragments are characterized by encoding a biologicallyactive portion of P39.5, e.g., an epitope. Generally, theseoligonucleotide fragments are at least 15 nucleotides in length.However, oligonucleotide fragments of varying sizes may be selected asdesired. Such fragments may be used for such purposes as performingpolymerase chain reaction (PCR), e.g., on a biopsied tissue sample. Forexample, useful fragments of P39.5 DNA and corresponding sequencescomprise sequences occurring between bp 793-816 of SEQ ID NO: 1 and bp59-85 of SEQ ID NO: 13. Other useful fragments are identified in FIG. 2.Fragments of 1-1 include sequences of SEQ ID NOS: 3, 4 and 5. Fragmentsof 3-1 include sequences of SEQ ID NOS: 6 and 7; fragments of 6-1include sequences of SEQ ID NOS: 8 and 9. A fragment of 9-1 includes thesequence of SEQ ID NO: 10. Fragments of 12-1 include the sequences ofSEQ ID NOS: 11 and 12. The complete sequences of 1-1,3-1, 6-1,9-1 and12-1 and other useful fragments may be readily obtained by one of skillin the art by resort to conventional DNA sequencing techniques appliedto the DNA deposited with the ATCC above.

[0054] The DNA sequences of SEQ ID NOS: 1 and 3-12 as well as thedeposited materials identified above permit one of skill in the art toreadily obtain the corresponding anti-sense strands of these DNAsequences. Further, using known techniques, one of skill in the art canreadily obtain additional genomic and cDNA sequences which flank theillustrated DNA sequences or the corresponding RNA sequences, asdesired. Similarly the availability of SEQ ID NOS: 1 and 3-12 and thedeposited materials of this invention permits one of skill in the art toobtain other species P39.5 analogs, B. garinii peptides, and fragmentsthereof, by use of the nucleic acid sequences of this invention asprobes in a conventional technique, e.g., polymerase chain reaction.Allelic variants of these sequences within a species (i.e., sequencescontaining some individual nucleotide differences from a more commonlyoccurring sequence within a species, but which nevertheless encode thesame protein or a protein with the same function) such as other variantsof P39.5 SEQ ID NO: 2, may also be readily obtained given the knowledgeof this sequence provided by this invention.

[0055] The present invention further encompasses nucleic acid sequencescapable of hybridizing under stringent conditions [see, J. Sambrook etal, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory (1989)] to the sequences of SEQ ID NO: 1, their anti-sensestrands, or biologically active fragments thereof. An example of ahighly stringent hybridization condition is hybridization at 2×SSC at65° C., followed by a washing in 0.1×SSC at 65° C. for an hour.Alternatively, an exemplary highly stringent hybridization condition isin 50% formamide, 4×SSC at 42° C. Moderately high stringency conditionsmay also prove useful, e.g., hybridization in 4×SSC at 55° C., followedby washing in 0.1×SSC at 37° C. for an hour. An alternative exemplarymoderately high stringency hybridization condition is in 50% formamide,4×SSC at 30° C.

[0056] According to the invention, the nucleic acid sequences may bemodified. Utilizing the sequence data of SEQ ID NOS: 1 and 3-12, it iswithin the skill of the art to obtain or prepare synthetically orrecombinantly other polynucleotide sequences, or modified polynucleotidesequences, encoding the full-length proteins or useful fragments of theinvention. Such modifications at the nucleic acid level include, forexample, modifications to the nucleotide sequences which are silent orwhich change the amino acids, e.g. to improve expression or secretion.Also included are allelic variations, caused by the natural degeneracyof the genetic code.

[0057] Also encompassed by the present invention are mutants of theP39.5 fragments and cassette string sequences 1-1,3-1, 6-1,7-1, 9-1 and12-1 provided herein. Such mutants include amino terminal, carboxyterminal or internal deletions, which substantially retain theantigenicity of the full-length P39.5 or other proteins or fragments.Such a truncated, or deletion, mutant may be expressed for the purposeof affecting the activity of the full-length or wild-type gene or genefragments.

[0058] These nucleic acid sequences are useful for a variety ofdiagnostic, prophylactic and therapeutic uses. Advantageously, thenucleic acid sequences are useful in the development of diagnosticprobes and antisense probes for use in the detection and diagnosis ofLyme disease by utilizing a variety of known nucleic acid assays, e.g.,Northern and Southern blots, polymerase chain reaction (PCR), and otherassay techniques known to one of skill in the art. The nucleic acidsequences of this invention are also useful in the production of P39.5proteins and homologs as well as other proteins of B. garinii.

[0059] The nucleotide sequences of the invention may be isolated byconventional uses of polymerase chain reaction or cloning techniquessuch as those described in conventional texts such as Sambrook et al,cited above. For example, the nucleic acid sequences of the antigen ofthis invention may be prepared or isolated from B. garinii DNA using DNAprimers and probes and PCR techniques. Alternatively, the antigen may beobtained from gene banks derived from B. garinii whole genomic DNA.These sequences, fragments thereof, modifications thereto and thefull-length sequences may be constructed recombinantly usingconventional genetic engineering or chemical synthesis techniques orPCR, and the like by utilizing the information provided herein.

[0060] B. Protein Sequences

[0061] The present invention also provides B. garinii proteins andpeptides including P39.5 polypeptides or proteins, and thepeptides/proteins represented by the designations P1-1, P3-1, P6-1,P7-1, P9-1 and P12-1 of this invention. These proteins are free fromassociation with other contaminating proteins or materials with whichthey are found in nature. Because the native P39.5 was fully extractedinto the detergent phase in a Triton-X114 extraction, it is likely alipoprotein. The P39.5 antigen has a relative molecular mass of 39,500daltons as measured by Western immunoblot (See Example 2 and FIG. 6). Inone embodiment, the invention provides a partial P39.5 [SEQ ID NO:2]polypeptide of about 354 amino acids, likely a lipoprotein, having apredicted relative molecular mass of about 37.7 kD (i.e., “the P7-1fragment”). This deduced amino acid sequence [SEQ ID NO: 2] is up to 22%identical to members of the variable major protein (Vmp) family of outersurface lipoproteins of Borrelia hermsii (see Example 6). It is about50% identical to the Vmp-like sequence (vls) VIsE of the B31 strain ofB. burgdorferi [J. -R. Zhang et al, cited above] and to other vlssequences of B. burgdorferi [i.e., Vls-6 Genbank no. U76406 (53%identity over 190 aa); VIsE Genbank no. U84553 (57% over 210 aa) andU84566 (58% over 209 aa) and U84555 (58% over 210 aa)].

[0062] VIsE is part of an antigen of B. burgdorferi that undergoesantigenic variation by a mechanism of recombination whereby a centralfragment of the expressed copy (VIsE) is recombined with fragments froma string of 15 “cassettes” located upstream from the expressed copy [J.Zhang, cited above]. Each cassette is on average 500 bp in length, andthe whole string occupies a stretch of DNA of about 8 kb. One of theremarkable features of this string of cassettes is that in B.burgdorferi B31, the cassettes are arranged in a nearly contiguous openreading frame interrupted only by a stop codon in cassette vls 11 andtwo frame shifts in cassettes vls 14 and vls 16. See, e.g., FIG. 6,which is a copy of a similar structure defined in Zhang et al, citedabove.

[0063] The present invention provides partial DNA and deduced amino acidsequences of several fragments which appear to be part of the cassettestring of B. garinii IP90. These fragments, which may include 7-1, arenamed 1-1,3-1, 6-1,9-1, and 12-1, and are between 1 and 2 kb in length.Each of them expresses a peptide (off of the lacZ promoter ofpBluescript) which is commensurate with the size of the insert and whichreacts with antibody from infected monkeys. None of the 5′ ends of thesefragments [SEQ ID NOS: 1, 3, 6, 8, 10 and 11] contains a hydrophobicleader or a signal peptidase II consensus sequence of the type that ischaracteristic of bacterial lipoproteins. These fragments must thereforebe part of the cassette string of IP90 and, like the cassette string ofB31, they are in-frame. These vls-like proteins are identified bypartial 5′ and 3′ sequences [see, e.g., SEQ ID NOS: 1, 3, 5 through 12].One of skill in the art using conventional techniques, such as PCR, mayreadily use the partial 5′ and 3′ sequences provided herein and the DNAsequences of (or DNA library of) B. garinii or the materials depositedwith the ATCC for each cassette string protein identified above, toidentify the complete sequences thereof. Such methods are routine andnot considered to require undue experimentation, given the informationprovided herein.

[0064] Antigens of this invention may be characterized by immunologicalmeasurements including, without limitation, western blot, macromolecularmass determinations by biophysical determinations, such asSDS-PAGE/staining, HPLC and the like, antibody recognition assays,T-cell recognition assays, MHC binding assays, and assays to inferimmune protection or immune pathology by adoptive transfer of cells,proteins or antibodies.

[0065] The proteinaceous P39.5 surface antigen of this invention (aswell as its naturally occurring variants or analogs in other species ofBorrelia or the other Vls-like proteins identified herein) may beisolated in the form of a complete intact protein, or a polypeptide orfragment thereof. In one embodiment, P39.5 is isolated by immunoblotprocedures according to its respective molecular mass, as describedbelow in Example 5. Such isolation provides the antigen in a formsubstantially free from other proteinaceous and non-proteinaceousmaterials of the microorganism and the tick vector. The moleculescomprising the Borrelia polypeptides and antigens of this invention maybe isolated from the spirochete and further purified using any of avariety of conventional methods including: liquid chromatography such asnormal or reversed phase, using HPLC, FPLC and the like, affinitychromatography (such as with inorganic ligands or monoclonalantibodies); size exclusion chromatography, immobilized metal chelatechromatography, gel electrophoresis; and the like. One of skill in theart may select the most appropriate isolation and purificationtechniques without departing from the scope of this invention.

[0066] Alternatively, the amino acid sequences of P39.5 or the other B.garinii proteins of this invention may be produced recombinantlyfollowing conventional genetic engineering techniques [see e.g.,Sambrook et al, cited above and the detailed description of making theproteins below].

[0067] i. Analogs/Modified Antigens

[0068] Also included in the invention are analogs, or modified versions,of the P39.5 protein or vls-like proteins P1-1, P3-1, P6-1, P7-1, P9-1and P12-1, provided herein. Typically, such analogs differ from thespecifically identified proteins by only one to four codon changes.Examples include polypeptides with minor amino acid variations from theillustrated partial amino acid sequence of, for example, P7-1 (SEQ IDNO: 2), in particular, conservative amino acid replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains and chemicalproperties. Also provided are homologs of the proteins of the inventionwhich are characterized by having at least 85% identity with SEQ ID NO:2or with the sequences of the vls-like proteins. Based on the sequenceinformation provided herein, one of skill in the art can readily obtainfull-length P7-1 or P7-1 homologs and analogs, as well as thefull-length vls-like 1-1, 3-1,6-1, 9-1 and 12-1 protein homologs andanalogs from other bacterial species.

[0069] An antigen of the present invention may also be modified toincrease its immunogenicity. For example, the antigen may be coupled tochemical compounds or immunogenic carriers, provided that the couplingdoes not interfere with the desired biological activity of either theantigen or the carrier. For a review of some general considerations incoupling strategies, see Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, ed. E. Harlow and D. Lane (1988). Useful immunogeniccarriers known in the art, include, without limitation, keyhole limpethemocyanin (KLH); bovine serum albumin (BSA), ovalbumin, PPD (purifiedprotein derivative of tuberculin); red blood cells; tetanus toxoid;cholera toxoid; agarose beads; activated carbon; or bentonite. Usefulchemical compounds for coupling include, without limitation,dinitrophenol groups and arsonilic acid.

[0070] The antigen may also be modified by other techniques, such asdenaturation with heat and/or SDS.

[0071] ii. Fragments/Deletion Mutants

[0072] Further encompassed by this invention are additional fragments ofthe P39.5 polypeptide, the P7-1 peptide or of the other vls-likeproteins identified herein. Such fragments are desirably characterizedby having a biological activity similar to that displayed by thecomplete protein, including, e.g., the ability to induce antibodies tothe causative agent of Lyme Disease. These fragments may be designed orobtained in any desired length, including as small as about 5-8 aminoacids in length. Such a fragment may represent an epitope of theprotein.

[0073] The internal repeats of P39.5 (see FIG. 2) indicate that thisprotein, like OspA, may undergo homologous recombinations in Borreliawhich may lead to mutants expressing shortened, partially-deletedversions of the gene. Thus, the P7-1 protein [SEQ ID NO:2] of theinvention may be modified to create deletion mutants, for example, bytruncation at the amino or carboxy termini, or by elimination of one ormore amino acids. Deletion mutants of P7-1 created by homologousrecombination of its repeat sequences (FIG. 2) are also encompassed bythis invention, as are the DNA sequences encoding them. In certaindeletion mutants, portions of the P39.5 coding region, described above,e.g., regions IIA, IIB, and B, are deleted.

[0074] Deletion mutants of the P39.5 antigens that, like P39.5 andunlike OspA, are expressed in vivo, will likely be killed upon infectionof the vertebrate host. Most OspA deletion mutants escape killing withanti-OspA antibody in the absence of complement, but are killed by thisantibody and complement acting together. Since anti-OspA antibody killsspirochetes in the tick midgut (where OspA is expressed, but complementis likely not active), OspA deletion mutants may escape the anti-Osp Aantibody elicited by the OspA vaccine. In contrast, since P39.5 isexpressed in vivo, where complement and anti-P39.5 antibody are bothpresent, P39.5 deletion mutants may not escape a P39.5 based vaccine.

[0075] Still other modified fragments of P39.5, P1-1, P3-1, P6-1, P7-1,P9-1, or P12-1 may be prepared by any number of now conventionaltechniques to improve production thereof, to enhance protein stabilityor other characteristics, e.g. binding activity or bioavailability, orto confer some other desired property upon the protein. Other usefulfragments of these polypeptides may be readily prepared by one of skillin the art using known techniques, such as deletion mutagenesis andexpression.

[0076] iii. Fusion or Multimeric Proteins and Compositions

[0077] The P39.5 protein of the present invention, or fragments of it,as well as the vls-like cassette string proteins or fragments thereof,may also be constructed, using conventional genetic engineeringtechniques as part of a larger and/or multimeric protein or proteincompositions. Antigens of this invention may be in a combination with B.burgdorferi outer surface proteins, such as OspA and OspB, or variousfragments of the antigens described herein may be in combination witheach other. In such combination, the antigen may be in the form of afusion protein. The antigen of the invention may be optionally fused toa selected polypeptide or protein, e.g. Borrelia antigens OspA and OspB,other Borrelia antigens, and proteins or polypeptides derived from othermicroorganisms. For example, an antigen or polypeptide of this inventionmay be fused at its N-terminus or C-terminus to OspA polypeptide, orOspB polypeptide or to a non-OspA non-OspB polypeptide or combinationsthereof. OspA and OspB polypeptides which may be useful for this purposeinclude polypeptides identified by the prior art [see, e.g.PCT/US91/04056] and variants thereof. Non-OspA, non-OspB polypeptideswhich may be useful for this purpose include polypeptides of theinvention and those identified by the prior art, including, the B.burgdorferi, flagella-associated protein and fragments thereof, other B.burgdorferi proteins and fragments thereof, and non-B. burgdorferiproteins and fragments thereof.

[0078] Still another fusion protein of this invention is provided byexpressing the DNA molecule formed by the P39.5 DNA sequence or afragment thereof fused to DNA fragments that are homologous (25-95%identity) to P39.5. One example of such a protein comprises the aminoacid sequence of SEQ ID NO: 2 to which is fused amino acid fragmentsthat are up to 95% identical to that sequence. These fragments may beinserted in any order and may contain repeated sequences such as thosedepicted in FIG. 2. These long strings of DNA that form a single openreading frame may be constructed from P39.5 homologs, including P39.5,or be derived from the naturally occurring long open reading frames,such as one or more of the vls cassette proteins. The vls cassetteproteins from B. garinii (e.g., the proteins designated P1-1, P3-1,P6-1, P9-1 and/or P12-1) may be fused to each other and/or inserted intothe P39.5 or P7-1 DNA sequence, thereby creating a large DNA moleculewhich expresses a protein which may stimulate a variety of antibodyspecificities.

[0079] These fusion proteins comprising multiple polypeptides of thisinvention are constructed for use in the methods and compositions ofthis invention. These fusion proteins or multimeric proteins may beproduced recombinantly, or may be synthesized chemically. They also mayinclude the polypeptides of this invention fused or coupled to moietiesother than amino acids, including lipids and carbohydrates. Further,antigens of this invention may be employed in combination with otherBorrelia vaccinal agents described by the prior art, as well as withother species of vaccinal agents derived from other viruses. Suchproteins are effective in the prevention, treatment and diagnosis ofLyme disease as caused by a wide spectrum of B. burgdorferi isolates.

[0080] A protein composition which may be a preferred alternative to thefusion proteins described above is a cocktail (i.e., a simple mixture)containing different P39.5 proteins or fragments, or different mixturesof the cassette string proteins of this invention. Such mixtures ofthese proteins or antigenic fragments thereof are likely to be useful inthe generation of desired antibodies to B. garinii.

[0081] iv. Salts

[0082] An antigen of the present invention may also be used in the formof a pharmaceutically acceptable salt. Suitable acids and bases whichare capable of forming salts with the polypeptides of the presentinvention are well known to those of skill in the art, and includeinorganic and organic acids and bases.

[0083] II. Methods of Making Antigens and Nucleic Acid Sequences of theInvention

[0084] A. Expression In Vitro

[0085] To produce recombinant P7-1 and/or other cassette string proteinsor other fragments of P39.5 of this invention, the DNA sequences of theinvention are inserted into a suitable expression system. Desirably, arecombinant molecule or vector is constructed in which thepolynucleotide sequence encoding the selected protein, e.g., P7-1, isoperably linked to a heterologous expression control sequence permittingexpression of the protein. Numerous types of appropriate expressionvectors are known in the art for protein expression, by standardmolecular biology techniques. Such vectors are selected from amongconventional vector types including insects, e.g., baculovirusexpression, or yeast, fungal, bacterial or viral expression systems.Other appropriate expression vectors, of which numerous types are knownin the art, can also be used for this purpose. Methods for obtainingsuch expression vectors are well-known. See, Sambrook et al, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor Laboratory,New York (1989); Miller et al, Genetic Engineering, 8:277-298 (PlenumPress 1986) and references cited therein.

[0086] Suitable host cells or cell lines for transfection by this methodinclude bacterial cells. For example, the various strains of E. coli(e.g., HB101, MC1061, and strains used in the following examples) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas, Streptomyces, and other bacilli and thelike are also be employed in this method.

[0087] Mammalian cells, such as human 293 cells, Chinese hamster ovarycells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived fromSwiss, Balb-c or NIH mice are used. Another suitable mammalian cell lineis the CV-1 cell line. Still other suitable mammalian host cells, aswell as methods for transfection, culture, amplification, screening,production, and purification are known in the art. [See, e.g., Gethingand Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman etal, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat.No. 4,419,446].

[0088] Many strains of yeast cells known to those skilled in the art arealso available as host cells for expression of the polypeptides of thepresent invention. Other fungal cells may also be employed as expressionsystems. Alternatively, insect cells such as Spodoptera frugipedera(Sf9) cells may be used.

[0089] Thus, the present invention provides a method for producing arecombinant B. garinii protein, e.g., P7-1 or other cassette stringproteins, which involves transfecting, e.g., by conventional means suchas electroporation, a host cell with at least one expression vectorcontaining a polynucleotide of the invention under the control of atranscriptional regulatory sequence. The transfected or transformed hostcell is then cultured under conditions that allow expression of theprotein. The expressed protein is recovered, isolated, and optionallypurified from the cell (or from the culture medium, if expressedextracellularly) by appropriate means known to one of skill in the art.

[0090] For example, the proteins are isolated in soluble form followingcell lysis, or extracted using known techniques, e.g., in guanidinechloride. If desired, the proteins or fragments of the invention areproduced as a fusion protein. Such fusion proteins are those describedabove. Alternatively, for example, it may be desirable to produce fusionproteins to enhance expression of the protein in a selected host cell,to improve purification, or for use in monitoring the presence of thedesired protein, e.g., P7-1, in tissues, cells or cell extracts.Suitable fusion partners for the proteins of the invention are wellknown to those of skill in the art and include, among others,β-galactosidase, glutathione-S-transferase, and poly-histidine.

[0091] B. Expression In Vivo

[0092] Alternatively, where it is desired that the P7-1 or othercassette string protein of the invention (whether full-length or adesirable fragment) be expressed in vivo, e.g., to induce antibodies, oras a DNA vaccine, an appropriate vector for delivery is readily selectedby one of skill in the art. Exemplary vectors for in vivo gene deliveryare readily available from a variety of academic and commercial sources,and include, e.g., adeno-associated virus [International patentapplication No. PCT/US91/03440], adenovirus vectors [M. Kay et al, Proc.Natl. Acad. Sci. USA, 91:2353 (1994); S. Ishibashi et al, J. Clin.Invest., 92:883 (1993)], or other viral vectors, e.g., variouspoxviruses, vaccinia, etc. Methods for insertion of a desired gene,e.g., P7-1, and obtaining in vivo expression of the encoded protein, arewell known to those of skill in the art.

[0093] III. Antibodies of the Invention

[0094] The present invention also provides antibodies capable ofrecognizing and binding the isolated, or modified, or multimericantigens of this invention, including antibodies derived from mixturesof such antigens or fragments thereof. These antibodies are useful indiagnosis of Lyme disease and in therapeutic compositions for treatinghumans and/or animals that test positive for, or, prior to testing,exhibit symptoms of, Lyme Disease. The antibodies are useful indiagnosis alone or in combination with antibodies to other antigens ofthis invention as well as antibodies to other known B. burgdorferiantigens. These antibodies are also useful in passive vaccinecompositions.

[0095] The antibodies of this invention are generated by conventionalmeans utilizing the isolated, recombinant or modified antigens of thisinvention, or mixtures of such antigens or antigenic fragments. Forexample, polyclonal antibodies are generated by conventionallystimulating the immune system of a selected animal or human with theisolated antigen or mixture of antigenic proteins or peptides of thisinvention, allowing the immune system to produce natural antibodiesthereto, and collecting these antibodies from the animal or human'sblood or other biological fluid.

[0096] For example, an antibody according to the invention is producedby administering to a vertebrate host the antigen or antigeniccomposition of this invention, e.g., P7-1. Preferably a recombinantversion of P7-1 (rP7-1) is used as an immunogen. A suitable polyclonalantibody against the P7-1 antigen kills IP90 spirochetes in vitro byantibody-dependent, complement-mediated killing (ADCK) regardless ofwhether the antibody was obtained by: (1) affinity purification using asimmunoabsorbant native P39.5 antigen of IP90 spirochetes (as separatedon a Western blot; see FIG. 6) and as source of antibody the antiserumgenerated during an infection of rhesus monkeys with JD1 spirochetes or,(2) by immunizing mice with recombinant P7-1 from IP90 (rP7-1).

[0097] Thus, an antibody of the invention is isolated by affinitypurifying antiserum generated during an infection of a vertebrateanimal, e.g., a rhesus monkey, with JD1 spirochetes, using asimmunoabsorbant the native P39.5 antigen of IP90, or one or more of thecassette string proteins identified herein. Similarly, an antibody ofthe invention is isolated by immunizing mice with a purified,recombinant antigen of this invention, or a purified, isolated P39.5 ofnative origin.

[0098] Monoclonal antibodies (MAbs) directed against P39.5 are alsogenerated. Hybridoma cell lines expressing desirable MAbs are generatedby well-known conventional techniques, e.g. Kohler and Milstein and themany known modifications thereof. Similarly desirable high titerantibodies are generated by applying known recombinant techniques to themonoclonal or polyclonal antibodies developed to these antigens [see,e.g., PCT Patent Application No. PCT/GB85/00392; British PatentApplication Publication No. GB2188638A; Amit et al., Science,233:747-753 (1986); Queen et al., Proc. Nat'l. Acad. Sci. USA,86:10029-10033 (1989); PCT Patent Publication No. WO9007861; Riechmannet al., Nature, 332:323-327 (1988); Huse et al, Science, 246:1275-1281(1988)a].

[0099] Given the disclosure contained herein, one of skill in the artmay generate chimeric, humanized or fully human antibodies directedagainst P39.5 or the cassette proteins, or antigenic fragments thereofby resort to known techniques by manipulating the complementaritydetermining regions of animals or human antibodies to the antigen ofthis invention. See, e.g., E. Mark and Padlin, “Humanization ofMonoclonal Antibodies”, Chapter 4, The Handbook of ExperimentalPharmacology, Vol. 113, The Pharmacology of Monoclonal Antibodies,Springer-Verlag (June, 1994).

[0100] Alternatively, the antigens are assembled as multi-antigeniccomplexes [see, e.g., European Patent Application 0339695, publishedNov. 2, 1989] or as simple mixtures of antigenic proteins/peptides andemployed to elicit high titer antibodies capable of binding the selectedantigen(s) as it appears in the biological fluids of an infected animalor human.

[0101] Further provided by the present invention are anti-idiotypeantibodies (Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 arespecific for the target to which anti-P39.5 antibodies of the inventionbind and Ab3 are similar to P39.5 antibodies (Ab1) in their bindingspecificities and biological activities [see, e.g., M. Wettendorff etal., “Modulation of anti-tumor immunity by anti-idiotypic antibodies.”In Idiotypic Network and Diseases, ed. by J. Cerny and J. Hiernaux J,Am. Soc. Microbiol., Washington D.C.: pp. 203-229, (1990)].

[0102] These anti-idiotype and anti-anti-idiotype antibodies areproduced using techniques well known to those of skill in the art. Suchanti-idiotype antibodies (Ab2) can bear the internal image of P39.5, thecassette proteins, or fragments thereof and are thus useful for the samepurposes as P39.5, the cassette proteins or the fragments.

[0103] In general, polyclonal antisera, monoclonal antibodies and otherantibodies which bind to the selected antigen (Ab1) are useful toidentify epitopes of P39.5 or the cassette proteins to separate P39.5(or the cassette proteins) and analogs thereof from contaminants inliving tissue (e.g., in chromatographic columns and the like), and ingeneral as research tools and as starting material essential for thedevelopment of other types of antibodies described above. Anti-idiotypeantibodies (Ab2) are useful for binding the same target and thus may beused in place of the original antigen, e.g., P39.5, to induce an immuneresponse. The Ab3 antibodies are useful for the same reason the Ab1 areuseful. Other uses as research tools and as components for separation ofP39.5 or the cassette proteins, from other contaminants, for example,are also contemplated for the above-described antibodies.

[0104] For use in diagnostic assays, the antibodies are associated withconventional labels which are capable, alone or in concert with othercompositions or compounds, of providing a detectable signal. Where morethan one antibody is employed in a diagnostic method, the labels aredesirably interactive to produce a detectable signal. Most desirably,the label is detectable visually, e.g. calorimetrically. A variety ofenzyme systems have been described in the art which will operate toreveal a calorimetric signal in an assay. As one example, glucoseoxidase (which uses glucose as a substrate) releases peroxide as aproduct. Peroxidase, which reacts with peroxide and a hydrogen donorsuch as tetramethyl benzidine (TMB) produces an oxidized TMB that isseen as a blue color. Other examples include horseradish peroxidase(HRP) or alkaline phosphatase (AP), and hexokinase in conjunction withglucose-6-phosphate dehydrogenase which reacts with ATP, glucose, andNAD+ to yield, among other products, NADH that is detected as increasedabsorbance at 340 nm wavelength. Other label systems that may beutilized in the methods of this invention are detectable by other means,e.g., colored latex microparticles [Bangs Laboratories, Indiana] inwhich a dye is embedded may be used in place of enzymes to formconjugates with the antibodies and provide a visual signal indicative ofthe presence of the resulting complex in applicable assays. Still otherlabels include fluorescent compounds, radioactive compounds or elements.Detectable labels for attachment to antibodies useful in diagnosticassays of this invention may be easily selected from among numerouscompositions known and readily available to one skilled in the art ofdiagnostic assays. The methods and antibodies of this invention are notlimited by the particular detectable label or label system employed.

[0105] IV. Diagnostic Methods and Assays

[0106] The present invention also provides methods of diagnosing Lymedisease. These diagnostic methods are useful for diagnosing humans oranimals exhibiting the clinical symptoms of, or suspected of having,Lyme disease.

[0107] In one embodiment, this diagnostic method involves detecting thepresence of naturally occurring anti-P39.5 antibodies which are producedby the infected human or animal patient's immune system in itsbiological fluids, and which are capable of binding to the antigens ofthis invention or combinations thereof. This method comprises the stepsof incubating a P39.5 antigen or a cassette string antigen of thisinvention with a sample of biological fluids from the patient.Antibodies present in the fluids as a result of B. burgdorferi infectionwill form an antibody-antigen complex with the antigen. Subsequently thereaction mixture is analyzed to determine the presence or absence ofthese antigen-antibody complexes. The step of analyzing the reactionmixture comprises contacting the reaction mixture with a labeledspecific binding partner for the antibody.

[0108] In a similar embodiment, this diagnostic method involvesdetecting the presence of naturally occurring anti-P1-1, anti-P3-1,anti-P6-1, anti P7-1, anti-P9-1, and/or anti-P 12-1 antibodies which areproduced by the infected human or animal patient's immune system in itsbiological fluids, and which are capable of binding to the antigens ofthis invention or combinations thereof. This method comprises the stepsof incubating one or preferably, a mixture, of these antigen(s) of thisinvention with a sample of biological fluids from the patient.Antibodies present in the fluids as a result of B. burgdorferi infectionwill form antibody-antigen complexes with the antigen(s). Subsequentlythe reaction mixture is analyzed to determine the presence or absence ofthese antigen-antibody complexes. The step of analyzing the reactionmixture comprises contacting the reaction mixture with a labeledspecific binding partner for the antibody.

[0109] In one embodiment of the method, purified antigen, fragment ormixture of antigens is electro- or dot-blotted onto nitrocellulosepaper. Subsequently, the biological fluid (e.g. serum or plasma) isincubated with the blotted antigen, and antibody in the biological fluidis allowed to bind to the antigen(s). The bound antibody is thendetected by standard immunoenzymatic methods.

[0110] In another embodiment of the method, latex beads are conjugatedto the antigen(s) of this invention. Subsequently, the biological fluidis incubated with the bead/protein conjugate, thereby forming a reactionmixture. The reaction mixture is then analyzed to determine the presenceof the antibodies.

[0111] In another embodiment, the diagnostic method of the inventioninvolves detecting the presence of the naturally occurring P39.5 orcassette string antigen(s) itself in its association with the Borreliapathogen in the biological fluids of an animal or human infected by thepathogen. This method includes the steps of incubating an antibody ofthis invention (e.g. produced by administering to a suitable humanand/or animal an antigen of this invention preferably conventionallylabelled for detection) with a sample of biological fluids from a humanor an animal to be diagnosed. In the presence of Borrelia infection ofthe human or animal patient, an antigen-antibody complex is formed(specific binding occurs). Subsequently, excess labeled antibody isoptionally removed, and the reaction mixture is analyzed to determinethe presence or absence of the antigen-antibody complex and the amountof label associated therewith.

[0112] Assays employing a protein antigen of the invention can beheterogenous (i.e., requiring a separation step) or homogenous. If theassay is heterogenous, a variety of separation means can be employed,including centrifugation, filtration, chromatography, or magnetism.

[0113] One preferred assay for the screening of blood products or otherphysiological or biological fluids is an enzyme linked immunosorbantassay, i.e., an ELISA. Typically in an ELISA, the isolated antigen(s) ofthe invention is adsorbed to the surface of a microtiter well directlyor through a capture matrix (i.e., antibody). Residual protein-bindingsites on the surface are then blocked with an appropriate agent, such asbovine serum albumin (BSA), heat-inactivated normal goat serum (NGS), orBLOTTO (a buffered solution of nonfat dry milk which also contains apreservative, salts, and an antifoaming agent). The well is thenincubated with a biological sample suspected of containing specificanti-B. burgdorferi antibody. The sample can be applied neat, or moreoften, it can be diluted, usually in a buffered solution which containsa small amount (0.1-5.0% by weight) of protein, such as BSA, NGS, orBLOTTO. After incubating for a sufficient length of time to allowspecific binding to occur, the well is washed to remove unbound proteinand then incubated with labeled anti-human immunoglobulin (αHuIg) orlabeled antibodies to other species, e.g., dogs. The label can be chosenfrom a variety of enzymes, including horseradish peroxidase (HRP),β-galactosidase, alkaline phosphatase, and glucose oxidase, as describedabove. Sufficient time is allowed for specific binding to occur again,then the well is washed again to remove unbound conjugate, and thesubstrate for the enzyme is added. Color is allowed to develop and theoptical density of the contents of the well is determined visually orinstrumentally.

[0114] Further, MAbs or other antibodies of this invention which arecapable of binding to the antigen(s) can be bound to ELISA plates. Inanother diagnostic method, the biological fluid is incubated on theantibody-bound plate and washed. Detection of any antigen-antibodycomplex, and qualitative measurement of the labeled MAb is performedconventionally, as described above.

[0115] Other useful assay formats include the filter cup and dipstick.In the former assay, an antibody of this invention is fixed to asintered glass filter to the opening of a small cap. The biologicalfluid or sample (5 mL) is worked through the filter. If the antigen ispresent (i.e., B. burgdorferi infection), it will bind to the filterwhich is then visualized through a second antibody/detector. Thedipstick assay involves fixing an antigen or antibody to a filter, whichis then dipped in the biological fluid, dried and screened with adetector molecule.

[0116] Other diagnostic assays can employ the antigen(s) or fragments ofthis invention as nucleic acid probes or an anti-sense sequences, whichcan identify the presence of infection in the biological fluid byhybridizing to complementary sequences produced by the pathogen in thebiological fluids. Such techniques, such as PCR, Northern or Southernhybridizations etc. are well known in the art.

[0117] It should be understood by one of skill in the art that anynumber of conventional protein assay formats, particularly immunoassayformats, or nucleic acid assay formats, may be designed to utilize theisolated antigens and antibodies or their nucleic acid sequences oranti-sense sequences of this invention for the detection of Borreliainfection in animals and humans. This invention is thus not limited bythe selection of the particular assay format, and is believed toencompass assay formats which are known to those of skill in the art.

[0118] V. Diagnostic Kits

[0119] For convenience, reagents for ELISA or other assays according tothis invention may be provided in the form of kits. Such kits are usefulfor diagnosing infection with Borrelia in a human or an animal sample.Such a diagnostic kit contains an antigen of this invention and/or atleast one antibody capable of binding an antigen of this invention, orthe nucleic acid sequences encoding them, or their anti-sense sequences.Alternatively, such kits may contain a simple mixture of such antigensor sequences, or means for preparing a simple mixture.

[0120] These kits can include microtiter plates to which the Borreliaantigen proteins or antibodies or nucleic acid sequences of theinvention have been pre-adsorbed, various diluents and buffers, labeledconjugates for the detection of specifically bound antigens orantibodies, or nucleic acids and other signal-generating reagents, suchas enzyme substrates, cofactors and chromogens. Other components ofthese kits can easily be determined by one of skill in the art. Suchcomponents may include polyclonal or monoclonal capture antibodies,antigen of this invention, or a cocktail of two or more of theantibodies, purified or semi-purified extracts of these antigens asstandards, MAb detector antibodies, an anti-mouse or anti-human antibodywith indicator molecule conjugated thereto, an ELISA plate prepared forabsorption, indicator charts for colorimetric comparisons, disposablegloves, decontamination instructions, applicator sticks or containers,and a sample preparator cup. Such kits provide a convenient, efficientway for a clinical laboratory to diagnose Borrelia infection.

[0121] VI. Therapeutic Compositions

[0122] The antigens, antibodies, nucleic acid sequences or anti-sensesequences of the invention, alone or in combination with other antigens,antibodies, nucleic acid sequences or anti-sense sequences may furtherbe used in therapeutic compositions and in methods for treating humansand/or animals with Lyme Disease. For example, one such therapeuticcomposition may be formulated to contain a carrier or diluent and one ormore of the anti-P7-1 or other anti-cassette string protein antibodiesof the invention. Suitable pharmaceutically acceptable carriersfacilitate administration of the proteins but are physiologically inertand/or nonharmful.

[0123] Carriers may be selected by one of skill in the art. Exemplarycarriers include sterile saline, lactose, sucrose, calcium phosphate,gelatin, dextran, agar, pectin, peanut oil, olive oil, sesame oil, andwater. Additionally, the carrier or diluent may include a time delaymaterial, such as glycerol monostearate or glycerol distearate alone orwith a wax. In addition, slow release polymer formulations can be used.

[0124] Optionally, this composition may also contain conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable ingredients which may be used in a therapeuticcomposition in conjunction with the antibodies include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

[0125] Alternatively, or in addition to the antibodies of the invention,other agents useful in treating Lyme disease, e.g., antibiotics orimmunostimulatory agents and cytokine regulation elements, are expectedto be useful in reducing or eliminating disease symptoms. Agents whichcan be used to suppress or counteract the immune suppressants releasedby the tick vector or the spirochete should act to assist the naturalimmunity of the infected human or animal. Thus, such agents may operatein concert with the therapeutic compositions of this invention. Thedevelopment of therapeutic compositions containing these agents iswithin the skill of one in the art in view of the teachings of thisinvention.

[0126] According to the method of the invention, a human or an animalmay be treated for Lyme Disease by administering an effective amount ofsuch a therapeutic composition. An “effective amount” may be betweenabout 0.05 to about 1000 μg/mL of an antibody of the invention. Asuitable dosage may be about 1.0 mL of such an effective amount. Such acomposition may be administered 1-3 times per day over a 1 day to 12week period. However, suitable dosage adjustments may be made by theattending physician or veterinarian depending upon the age, sex, weightand general health of the human or animal patient. Preferably, such acomposition is administered parenterally, preferably intramuscularly orsubcutaneously. However, it may also be formulated to be administered byany other suitable route, including orally or topically.

[0127] VII. Vaccine Compositions

[0128] None of the potential problems of the prior art arise with avaccine based on an antigen which is expressed in the vertebrate host.An improved surface-antigen-based vaccine for the prevention of Lymedisease in humans and other animals is provided by the inclusion of theP39.5 antigen of this invention, and a pharmaceutically acceptablecarrier or diluent. This vaccine composition may contain one or more ofthe isolated, recombinant, modified or multimeric forms of the P39.5antigen of the invention, or mixtures thereof. Similarly, salts of theantigenic proteins may be employed in such compositions.

[0129] Another embodiment of a composition of the present invention isbased on the other cassette string antigens, i.e., P1-1, P3-1, P6-1,P7-1, P9-1 and P12-1. The inventor proposes that the mechanism ofantigenic variation to which the vlsE antigen is subjected indicatesthat this antigen is crucially important for spirochetal survival in thevertebrate host. According to this invention, a novel vaccine methodinvolves circumventing the spirochetal defense mechanism by immunizing ahost with a large fraction of the epitopes that are expressed on thecassette string that is the very source of the variation. This is madepossible by the unique fact that most of this cassette string is inframe. The inventors have demonstrated this concept, as described in theExamples below, by immunizing a host with the cassette string antigen,P7-1, and inducing antibody that kills the spirochetes. Thus, accordingto this invention, one method of vaccination thus involves administeringto the host several or all of the cloned and purified proteins describedabove, which are derived from antigenically dissimilar sections of theIP90 cassette string. While a variety of immunogenic compositions may beprepared from such proteins, it is presently preferred that a simplemixture of two or more of the cassette string proteins or peptidefragments thereof be employed for this purpose.

[0130] Combinations of the antigen(s) of this invention with otherantigens of B. burgdorferi, such as the OspA and OspB proteins, BmpA, B,C or D proteins, or fragments thereof are also encompassed by thisinvention.

[0131] Exemplary carriers are as described above for therapeuticcompositions. Optionally, the vaccine composition may further containadjuvants, preservatives, chemical stabilizers, or other antigenicproteins. Typically, stabilizers, adjuvants, and preservatives areoptimized to determine the best formulation for efficacy in the targethuman or animal. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallade, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.

[0132] One or more of the above described vaccine components may beadmixed or adsorbed with a conventional adjuvant. The adjuvant is usedto attract leukocytes or enhance an immune response. Such adjuvantsinclude, among others, Ribi, mineral oil and water, aluminum hydroxide,Amphigen, Avridine, L 121/squalene, D-lactide-polylactide/glycoside,pluronic plyois, muramyl dipeptide, killed Bordetella, and saponins,such as Quil A. In addition, a vaccine composition of the invention mayfurther comprise other, non-B. burgdorferi antigens, including,Bordetella bronchiseptica, canine parvovirus, canine distemper, rabies,Leptosporidia, canine coronavirus, and canine adenovirus. Other vaccinalantigens originating from other species may also be included in thesecompositions, e.g., feline coronavirus, etc.

[0133] The invention thus also encompasses a prophylactic methodentailing administering to an animal or human an effective amount ofsuch a composition. The P39.5 and/or cassette string protein antigeniccompositions are administered in an “effective amount”, that is, anamount of antigen that is effective in a route of administration toprovide a vaccinal benefit, i.e., protective immunity. Suitable amountsof the antigen can be determined by one of skill in the art based uponthe level of immune response desired. In general, however, the vaccinecomposition contains between 1 ng to 1000 mg antigen, and morepreferably, 0.05 μg to 1 mg per mL of antigen. Suitable doses of thevaccine composition of the invention can be readily determined by one ofskill in the art. Generally, a suitable dose is between 0.1 to 5 mL ofthe vaccine composition. Further, depending upon the human patient orthe animal species being treated, i.e. its weight, age, and generalhealth, the dosage can also be determined readily by one of skill in theart.

[0134] In general, the vaccine will be administered once on a seasonalbasis. Each tick season, usually in the spring, a booster should beadministered. The vaccine may be administered by any suitable route.However, parenteral administration, particularly intramuscular, andsubcutaneous, is the preferred route. Also preferred is the oral routeof administration. Routes of administration may be combined, if desired,or adjusted.

[0135] Further, the vaccine may be a DNA vaccine, which includes theP7-1 DNA sequence or a fragment thereof, another cassette stringprotein, or a fragment thereof, optionally under the control ofregulatory sequences. Thus, the antigen-encoding DNA may be carried in avector, e.g., a viral vector. Generally, a suitable vector-basedtreatment contains between 1×10⁻³ pfu to 1×10¹² pfu per dose. However,the dose, timing and mode of administration of these compositions may bedetermined by one of skill in the art. Such factors as the age, andphysical condition of the vaccinate may be taken into account indetermining the dose, timing and mode of administration of theimmunogenic or vaccine composition of the invention.

[0136] VIII. Drug Screening and Development

[0137] The proteins, antibodies and polynucleotide sequences of thepresent invention may also be used in the screening and development ofchemical compounds or proteins which have utility as therapeutic drugsor vaccines for the treatment or diagnosis or prevention of LymeDisease. As one example, a compound capable of binding to P39.5 andpreventing its biological activity may be a useful drug component forthe treatment or prevention of Lyme Disease. The methods describedherein may also be applied to fragments of P39.5. Similarly, a compoundcapable of binding to a cassette string protein, or fragment thereof andpreventing its biological activity may be a useful drug component forthe treatment or prevention of Lyme Disease.

[0138] Suitable assay methods may be readily determined by one of skillin the art. Where desired, and depending on the assay selected, theselected antigen(s), e.g., P39.5, may be immobilized directly orindirectly (e.g., via an anti-P39.5 antibody) on a suitable surface,e.g., in an ELISA format. Such immobilization surfaces are well known.For example, a wettable inert bead may be used. Alternatively, theselected antigen, e.g., P39.5, may be used in screening assays which donot require immobilization, e.g., in the screening of combinatoriallibraries. Assays and techniques exist for the screening and developmentof drugs capable of binding to an antigen of this invention, e.g.,P39.5. These include the use of phage display system for expressing theantigenic protein(s), and using a culture of transfected E. coli orother microorganism to produce the proteins for binding studies ofpotential binding compounds. See, for example, the techniques describedin G. Cesarini, FEBS Letters, 307(1):66-70 (July 1992); H. Gram et al.,J. Immunol. Meth., 161:169-176 (1993); C. Summer et al., Proc. Natl.Acad. Sci., USA, 89:3756-3760 (May 1992), incorporated by referenceherein.

[0139] Other conventional drug screening techniques may be employedusing the proteins, antibodies or polynucleotide sequences of thisinvention. As one example, a method for identifying compounds whichspecifically bind to a protein of this invention, e.g., P39.5, caninclude simply the steps of contacting a selected P39.5 protein with atest compound to permit binding of the test compound to P39.5; anddetermining the amount of test compound, if any, which is bound to theP39.5 protein. Such a method may involve the incubation of the testcompound and the P39.5 protein immobilized on a solid support. Similarmethods may be employed for one or more of the cassette string proteins.

[0140] Typically, the surface containing the immobilized ligand ispermitted to come into contact with a solution containing the proteinand binding is measured using an appropriate detection system. Suitabledetection systems include the streptavidin horse radish peroxidaseconjugate, direct conjugation by a tag, e.g., fluorescein. Other systemsare well known to those of skill in the art. This invention is notlimited by the detection system used.

[0141] Another method of identifying compounds which specifically bindto P39.5 or another protein of this invention can include the steps ofcontacting the protein, e.g., P39.5, immobilized on a solid support withboth a test compound and the protein sequence which is a receptor forP39.5 to permit binding of the receptor to the P39.5 protein; anddetermining the amount of the receptor which is bound to the P39.5protein. The inhibition of binding of the normal protein by the testcompound thereby indicates binding of the test compound to the P39.5protein. Similar methods may be employed for one or more of the cassettestring proteins.

[0142] Thus, through use of such methods, the present invention isanticipated to provide compounds capable of interacting with P39.5 orportions thereof, and/or the cassette string protein(s) or portionsthereof and either enhancing or decreasing the protein's biologicalactivity, as desired. Such compounds are believed to be encompassed bythis invention. Further wherever, above or below, the P39.5 protein ismentioned, it may be substituted with P7-1 or one or more of the othercassette string proteins.

[0143] The following examples illustrate the preferred methods forobtaining protein antigens of the invention and preparing the assays andcompositions of the invention. Significantly, these examples indicatethat the P39.5 antigen of this invention is useful for diagnosis andprophylaxis against Lyme disease and may improve Lyme serology. Theseexamples are illustrative only and do not limit the scope of theinvention.

EXAMPLE 1 Borrelia Bacterial Strains and Antibodies

[0144] A. Bacterial Strains

[0145] The JD1 strain of B. burgdorferi sensu stricto [J. Piesman etal., J. Clin. Microbiol., 25:557-558 (1987) and T. G. Schwan et al, J.Clin. Microbiol., 27:1734-1738 (1989)] was obtained from the Center forDisease Control and Prevention and kept in frozen stocks of low passage(<7). B31 is also a strain of B. burgdorferi sensu stricto obtained fromthe CDC and kept as above. IP90 is a strain of B. garinii which was alsofurnished by the CDC.

[0146]B. burgdorferi organisms were cultured at 34° C. in a BSK-Hculture medium (Sigma Chemical Co., St. Louis, Mo).

[0147] B. Monkey Antibodies

[0148] Live spirochetes were incubated with serum antibody from rhesusmonkeys that had been infected with B. burgdorferi strain JD1 by thebite of I. scapularis nymphs. Unbound antibodies were washed off thespirochetes and bound antibodies were removed with a low pH buffer.Monkey antibodies were obtained from three sources:

[0149] a) from rhesus monkeys infected with B. burgdorferi strain JD1 byneedle inoculation,

[0150] b) from rhesus monkeys infected with B. burgdorferi strain JD1 byexposure of the animals to JD1-infected nymphs of Ixodes scapularis,

[0151] c) by affinity purification on live spirochetes using as startingmaterial antiserum that was collected from tick-inoculated animals, asfollows: A volume of 800 μl of diluted ({fraction (1/40)} in BSK-Hmedium), heat-inactivated serum samples pooled from 3 tick-inoculatedanimals are incubated with 1×10⁹ total live bacteria at room temperaturefor 30 minutes. After the incubation, the samples were centrifuged at13,000×g for 15 minutes at 4° C. The supernatant was then readsorbed twomore times as described above. The bacterial pellet that was recoveredafter the first adsorption was washed three times with BSK-H to removeunbound antibodies. After the last wash, the pellet was resuspended in400-500 μl of 0.2 M glycine-HCl, pH 2.2, 0.5 M NaCl, and centrifuged.The supernatant was recovered and the pH brought to 7.00 by the additionof 2.0 M Tris base.

EXAMPLE 2 Preliminary Identification of P39.5 of IP90

[0152] The antibodies of Example 1, section B were reacted with Westernblots of whole extracts from JD1, B31 and IP90 spirochetes. Westernblots were performed as follows: Antigen preparations wereelectrophoresed in 15% acrylamide mini gels (10×10×0.1 cm) with a 5%acrylamide stacking gel. Twenty μl of lysate containing 7×10⁸solubilized bacteria or 25 μg of protein (measured by OD at 280 nm) weredispensed per track (the whole preparative track is equal to 16 singletracks, therefore 400 μg protein were loaded onto each preparative gel).Electrophoresis was performed using a mini-gel apparatus (IntegratedSeparation Systems, Hyde Park, Mass.) at a constant current of 23 mA,with the buffers of U. Laemmli, Nature, 227:680-685 (1970). Forimmunoblotting, the proteins from the polyacrylamide gels wereelectrotransferred to nitrocellulose paper (Schleicher and Schuell,Keene, N.H.) overnight at a constant voltage of 22 V in a Mighty Smalltransfer unit (Hoeffer Scientific Instruments, San Francisco, Calif.) asdescribed by H. Towbin et al, Proc. Natl. Acad. Sci. USA, 76:4350-4354(1979). Efficiency of transfer was assessed by staining part of thenitrocellulose with colloidal gold (Integrated Separation Systems).Nitrocellulose membranes were blocked with 3% fat-free powdered milk(Carnation) prepared in PBS containing 0.05% Tween-20 (IntegratedSeparation Systems) (PBS-T) for two hours at room temperature.

[0153] After the blocking step, the membranes were mounted in aMiniblotter 45 (Immunetics, Cambridge, Mass.) according to themanufacturer's instructions, and 110 μl of each serum sample diluted{fraction (1/50)} with PBS-T were introduced into the Miniblotter'schannels and allowed to interact with the nitrocellulose membrane forhour at room temperature on a rocking platform. After the incubation,the manifold system was used to wash the membranes with PBS-T. At thispoint the Miniblotter was disassembled and the blot was taken out. Therest of the incubation steps were performed in small trays. After thewash, the membranes were incubated for 1 hour with biotinylatedanti-human IgM (μ-chain-specific) and IgG (γ-chain-specific) antibodies(Vector Laboratories, Burlingame, Calif.) diluted {fraction (1/200)} inPBS-T. Biotinylated antibodies were probed with an avidin/biotinylatedhorseradish peroxidase complex (Vector) prepared according to themanufacturer's instructions. The reagent 4-chloro-1-naphthol (Sigma) wasused as a chromogen. The color reaction was stopped by washing themembranes with distilled water.

[0154] A Western blot (FIG. 6) was prepared of lysates from spirochetesof B. burgdorferi strains JD1 and B31 and B. garinii strain IP 90developed with serum from monkeys needle-inoculated and tick-inoculatedwith the JD1 spirochetes, and antibodies from the latter serum affinitypurified off of whole live JD1 spirochetes. The affinity purifiedantibody recognized four antigens on Western blots of whole lysates fromspirochetes of B. burgdorferi JD1. The antigens were named P1 (Relativemolecular mass (Mr) 39-40,000), P2 (Mr 35-37,000), P3 (Mr 22-24,000) andP4 (Mr 18-19,000). These antigens were also recognized, as expected, byserum samples from needle-inoculated animals and by serum fromtick-inoculated monkeys. In addition, this Western blot indicated thatthe affinity-purified antibodies recognized what appeared to be P1, P2,and P4 on B31 spirochetes and what appeared to be P1 (but with aslightly higher relative molecular mass) in B. garinii as well as anadditional antigen of higher relative molecular mass. These two latterantigens were also exclusively recognized by the sera from both needle-and tick-inoculated animals. P1 was eventually identified as P39, alsoknown as BmpA. The similar antigen present on IP90 spirochetes wastentatively identified as P39.5 and was shown on the Western blotdiscussed above.

EXAMPLE 3 Antibody-Dependent Complement-Mediated Killing of JD1, B31 andIP90 Spirochetes

[0155] Serum from tick-inoculated monkeys was employed in an ADCK assayas follows. Frozen samples of B. burgdorferi are quickly thawed at 37°C., cultured until they have reached mid-log phase (approximately 3days, 1-2×10⁷ spirochetes/ml), centrifuged at 8,000×g for 20 minutes,resuspended in BSK-H medium, and counted. The ADCK assay was carried outin duplicate in 96-well tissue culture plates (Costar). A total of5-6×10⁵ spirochetes in 25 μl of BSK-H medium was added to each wellcontaining 50 μl of heat-inactivated (56° C., 30 minutes) serum samplesdiluted to 1:10 in the same medium. The plates were incubated at 34° C.under a gas mixture of 3% CO₂, 5% O₂ and the balance of N₂ for 20minutes before the addition of 25 μl of complement (normal monkeyserum). After 18-24 hours of incubation under the same conditions, thetotal numbers of dead (nonmotile) and live (motile) bacteria werequantified under a dark-field microscope. Equivalence betweennon-motility and death in a similar assay has been determined before [M.Aydintug et al., cited above]. Killing was considered significant if themean % of dead spirochetes exceeds three times the value of the mean %killing observed in the presence of normal serum.

[0156] ADCK was performed with spirochetes from B. burgdorferi strainsJD1 and B31 and B. garinii strain IP90, using serum from animals thatwere tick inoculated with JD1 spirochetes. As shown in Example 2, thisserum recognized only two antigens on IP90 blots, one of a relativemolecular mass similar to that of P1, and one of higher relativemolecular mass. As is shown in FIG. 1, all three spirochetal strainswere killed by the serum, indicating that at least one of the antigensshown in the IP90 Western blot was the target of ADCK.

EXAMPLE 4 Identification of P39.5

[0157] The identity of the putative BmpA (P39) band seen on the IP90Western blots was investigated. A Western blot of lysates from JD1 andIP90 was developed with anti-flagellin monoclonal antibody (Mab);anti-P39 Mab; anti-P39 polyclonal antibody; anti-P35 Mab; anti BmpDpolyclonal; and sera from two monkeys tick-inoculated with JD1spirochetes. In the comparative Western blot analysis of JD1 and IP90antigens that were run on the same gel, both antigen extracts reactedwith the anti-flagellin Mab H9724. However, an anti-BmpA Mab reactedonly with JD1 BmpA, and serum from the monkey immunized with recombinantBmpA from JD1 reacted strongly with the JD1 blot, as expected, but veryfaintly with the BmpA antigen from IP90. Moreover, serum samplesobtained from two JD1-infected monkeys reacted with the same twoantigens as before on the IP90 blot and occasionally yielded additional(more faint) bands of higher molecular mass. The band of lowestmolecular mass of the two corresponds in fact to a larger molecule thanthe BmpA from IP90, as identified with the anti-BmpA polyclonalantibody. The higher molecular mass band likely corresponds toflagellin, as it had the same molecular mass as the band of IP-90 thatreacted with the MAb H9724.

[0158] A monoclonal antibody raised against P35 from JD1 reacted withthis molecule on the JD1 blot but not on the IP90 blot, whereas a mousepolyclonal antiserum raised against JD1 BmpD reacted with BmpD of bothJD1 and IP90, although the BmpD band of IP90 was very faint. Thus, theidentity of the IP90 antigen that could be the target of the ADCK wasnot BmpA, BmpD or P35. It was referred to hereafter as P39.5.

[0159] A Western blot was then developed of lysates of JD1 and IP90spirochetes incubated with serum from a monkey tick-inoculated withJD1); monkey polyclonal antiserum to P39; mouse Mab H9724 to flagellin;monkey anti-P39.5 antibody obtained by affinity purification from aserum sample from a monkey tick-inoculated with JD1; and mouseanti-recombinant P7-1 (rP7-1). To identify P39.5 on Western blots of JDlysates, the anti-P39.5 antibody elicited in JD1-infected monkeys wasaffinity purified using nitrocellulose strips that had P39.5 from IP90attached to them. As expected, this antibody reacted with the P39.5antigen on a Western blot of IP90 lysate but, surprisingly, failed toreact with the JD1 blot. This result implied that P39.5 of JD1 waseither not expressed in vitro, was sparsely expressed as to beundetectable by the affinity-purified anti-P39.5 antibody on the JD1blots, or was expressed in vitro by JD1 in a manner that was notcross-reactive antigenically with the antibody affinity purified off ofthe P39.5 of IP90.

[0160] Obviously, JD1 spirochetes had to express P39.5 in vivo and inamounts sufficiently high as to be immunogenic, since otherwise theanti-P39.5 antibody that was affinity purified would not have beenelicited in the first place. In the above described Western blot, theposition of BmpA was indicated by its reaction with the anti-BmpApolyclonal antiserum and that of flagellin by the reaction of thismolecule with Mab H9724.

[0161] A new antigen that is abundantly expressed in vitro by the B.garinii strain IP90 and in vivo by the B. burgdorferi sensu strictostrain JD1 was thus identified. This antigen, P39.5, was the target inthe ADCK of IP90 spirochetes.

EXAMPLE 5 Cloning of P39.5

[0162] A. Cloning in Bacteriophage

[0163] A library of randomly-sheared total DNA from B. garinii IP90 wasconstructed in the λZAPII bacteriophage vector [Stratagene, La Jolla,Calif.] and screened with a pool of plasma collected from rhesus monkeysinfected with the JD1 strain of B. burgdorferi. The plasma samples usedfor the pool were selected such that they contained antibody thatrecognized only P39.5, the putative flagellin and the 1 or 2 additionalunidentified higher molecular mass faint bands seen on some IP90 Westernblots.

[0164] After several rounds of screening, eleven clones were rescuedinto the pBluescript phagemid [Stratogene] the recombinant plasmids werepurified and used to transform cells of the SURE strain of E. coli.Several transformants were selected from each original clone, thepresence of the insert was confirmed, and one such transformant fromeach clone was grown, induced for expression, lysed and analyzed byWestern blot with the original plasma pool. The eleven cloned fragmentshybridized to each other by dot-blot hybridization.

[0165] One of the eleven clones (named 7-1) was selected forover-expression and purification on the basis of the strong reactivityof the expressed protein with the plasma antibodies. The 7-1 insert was950 bp in length.

[0166] The identity of the expressed protein was confirmed asantigenically identical to, or cross-reactive with, P39.5, by showingthat antibody from the original plasma sample that was affinity purifiedusing the clone as immunoabsorbant reacted with P39.5 on a Western blotof B. garinii lysate. A Western blot was developed of lysates from IP90spirochetes reacted with plasma from a monkey infected with JD1spirochetes; antibody from the same plasma affinity purified with therecombinant antigens as expressed by B. garinii clone 1-1 and clone 7-1.Reactivity of the IP90 lysate with the monkey plasma pool wasdemonstrated in the blot. Reactivity of the IP90 lysate with theantibody affinity purified with the recombinant 1-1 protein was shown,and with the antibody affinity purified with the recombinant 7-1protein.

[0167] A partial DNA sequence for P7-1 was obtained [SEQ ID NO: 1].About 950 bp were derived from clone 7-1. In addition, about 140 kb ofthe upstream regions were obtained from clone 14 [SEQ ID NO: 13]. TheDNA fragment formed by the 5′ SEQ ID NO: 13 sequence and the SEQ ID NO:1 sequence is 1189 bp in length, which is depicted in FIG. 2. Itencompasses a single open reading frame which encodes a deduced proteinof 37.7 kDa. Its high alanine content results in this rather lowmolecular mass. Since the average molecular mass of this protein's aminoacids is 95, there are about 57 bp of the complete coding regionmissing. Since no hydrophobic leader sequence was observed and sinceP39.5 has the solubility properties of lipoproteins (see below), itfollows that P7-1 is antigenically cross-reactive with P39.5, but is notthe complete P39.5 itself.

[0168] Data other than the deduced amino acid sequence of P7-1 [SEQ IDNOS: 14 and 2] suggests that P39.5 is a lipoprotein. First, the nativeform of P39.5 present in whole-cell extracts of IP90 spirochetes wasfully extractable, i.e., partitions into, the detergent phase in aTriton-X114 phase separation experiment. Second, most of its sequence iscomposed of hydrophilic domains, very much like OspA, yet it must beexpressed on the outer surface, since it is targeted by antibody. Third,its % identity with several members of the Vmp family of Borreliahermsii lipoproteins is considerable, e.g., 22% with Vmp4, 19% withVmp23, 18% with Vmp17, and 17% with Vmp21. These sequences are availableon the GENBANK database.

[0169] An interesting feature of its nucleotide sequence is the presenceof several internally repeated regions, shown in FIG. 2. Blocks IA andIB are 70% identical. Block IIA is as much as 91% identical to IIB, andblocks A, B and C are between 84 and 90% identical. The only internallyunique fragment is that denoted by the black block. Clearly, such amolecule will be prone to undergoing homologous recombinations of thesame kind that occur between and within the OspA and OspB genes, whichhave regions of homology [P. Rosa et al, cited above]. However, suchpotential escape mutants may not escape the combined action of antibodyand complement, as demonstrated by reference to the case of OspA [Soleet al, cited above].

[0170] B. Expression and Purification of Recombinant P7-1 with theQiaexpress™ System

[0171] The Qiaexpress™ system from Qiagen, Inc. (Chatsworth, Calif.)takes advantage of the high affinity of six consecutive histidineresidues for divalent cations such as Ni. The latter is conjugated tonitrilo-tri-acetic acid (NTA), which in turn is linked to agarose. Sincethe affinity tag is very small (minimally 6 residues) it may be left inthe fusion protein without serious consequences with regard to theprotein antigenic properties. The 6×His affinity tag may be incorporatedeither at the N- or C-terminus. A range of vectors (pQE vectors) areavailable for constructing both types of fusion.

[0172] Recombinants were initially screened for the presence of expectedinserts in the plasmids. This was followed by a Western blot to confirmprotein expression in these recombinants. Westerns blots were developedeither with serum from control mice infected with the spirochetes of thecorresponding strain or with a mouse antibody that specifically detectsthe MRGS(His)6 epitope (Qiagen), which was the affinity tag sequencepresent in some of the pQE vectors [for N-terminal fusions]. An equallysensitive alternative was to use Ni-NTA-alkaline phosphatase conjugate,which was also available from Qiagen.

[0173] The E. coli cells containing the fusion construct were grown tomid-log phase, induced with 0.3 mM IPTG for a period of three hours,pelleted, washed once with Sonication buffer [50 mM NaPO₄, pH 8.0, 300mM NaCl] and stored overnight at −20° C. On the next day, cells wereresuspended in Sonication buffer and sonicated on ice using 15-secondpulses for a total of 2 minutes. Immediately following sonication theserine protease inhibitor PMSF (phenyl-methyl-sulfonide fluoride) wasadded to prevent degradation by such proteases. The cell debris was spundown, and the supernatant transferred to a fresh tube. In the mean time,the Ni-NTA resin was washed once with 10 column volumes (cv) of theSonication buffer. The fusion protein was allowed to bind to the Ni-NTAresin in a tube for 1 hour. The resin was spun down and the supernatantdiscarded. The resin was resuspended in 10 cv of Sonication buffer,poured into a column and washed 3 times with 10 cv of the same bufferfollowed by 310-cv washes with Wash buffer [50 mM NaPO₄, pH 6.3, 300 mMNaCl]. Finally, the fusion protein was eluted with 10 cv of Elutionbuffer [50 mM NaPO₄, pH 4.5, 300 mM NaCl] and fractions collected.Fractions were assayed for protein and the fractions that contain thehighest protein concentration pooled and neutralized to pH 7.0 withdibasic NaPO₄. The protein concentration in the pooled sample wascalculated and an aliquot run on a SDS-PA gel and stained with silver tocheck purity. Typical yields were about 4-5 mg/liter of culture.

EXAMPLE 6 In Vitro Protective Potential of P7-1 in Monkeys

[0174] Anti-P7-1 antibody was purified from the same monkey plasma poolused to screen the DNA library, by absorbing and acid-eluting it off ofnitrocellulose strips that contained the recombinant P7-1 protein(rP7-1) as expressed from clone 7-1. The monospecificity of the antibodywas confirmed on a Western blot as described in Examples 2 and 4, andthe antibody was used in ADCK as described in Example 3.

[0175] The fraction of dead IP90 spirochetes after a 24 hour incubationwith the affinity purified anti-P7-1 antibody was 55% (FIG. 3, bar 2).The antibody was reconstituted at a concentration equivalent to a 1:10dilution of its concentration in plasma. This killing rate wascomparable to that observed with a 1:10 dilution of the same plasma(67%, FIG. 3, bar 1). Complement was essential to effect killing, asonly 12% of the spirochetes was killed in its absence (FIG. 3, bar 3).Killing in the presence of monkey complement alone was slightly higherthan usual, in that the value was 25% (FIG. 3, bar 4) whereas the valuemost frequently obtained was 10-15%, albeit with JD1 spirochetes [M.Aydintug et al, Infect. Immun., 62:4929-4937 (1994)]. In BSK-H mediumalone 12% of the spirochetes died (bar 5).

[0176] These results indicated that antibody to the recombinant P7-1 (afragment of P39.5) antigen of IP90, elicited in monkeys during thecourse of a natural infection with JD1 spirochetes, could kill IP90spirochetes by ADCK.

[0177] These experiments demonstrated the protective potential of P7-1and formed the basis of selection of this antigen as a vaccinecandidate.

EXAMPLE 7 Protective Potential of P7-1 in Mice

[0178] This example shows that mice may be directly immunized with therecombinant P7-1. The mouse antibodies generated by immunization withrP7-1 and Ribi adjuvant were able to kill up to 60% of IP90 spirochetesin vitro, by antibody-dependent, complement-mediated killing (ADCK). Toassess the protective potential of P7-1 more directly, the DNA fragmentof clone 7-1 was subcloned in the pQE as described in Example 5, and theexpressed protein was purified in milligram quantities and used toimmunize C3H/HeJ mice. Mice were given four injections of 30 μg each ofthe recombinant P7-1 in 0.2 ml of Ribi R-700 adjuvant (Ribi ImmunoChemResearch, Inc., Hamilton, Mont.). The R-700 formulation of 0.25 mg/mlMPL™, 0.25 mg/ml synthetic trehalose dicorynomycolate, 20 μl/ml Squalene(hexamethyl-tetracosahexane) and 2 μl/ml monooleate (Tween 80).Injections were given intraperitoneally, three weeks apart. Two weeksafter the last immunization, mice were bled, specificity of the elicitedantibody was confirmed by Western blot using whole cell extracts of IP90spirochetes as antigen, and serum samples were pooled.

[0179] Serum from mice immunized with the Ribi adjuvant alone recognizedno antigens on IP90 blots. Serum from mice immunized with rP7-1 and Ribirecognized, as expected, the P39.5 band on IP90 lysates, but also aweaker band slightly above the 41 kDa flagellin band, and a highermolecular mass band as well. Interestingly, this same mouse antibodyrecognized a 41 kDa band on Western blots of JD1 lysates, and also aslightly higher molecular mass antigen.

[0180] This result differed from that obtained with affinity-purifiedanti-rP7-1 antibody from monkeys in the preceding example and wasprobably due to the fact that the mouse antibody's affinity was allowedto mature for much longer (12 weeks) than that of the monkey antibody(4-5 weeks). As a consequence, its binding affinity was higher but itsspecificity was lower and it can bind to crossreactive but nonidenticalepitopes.

[0181] After an additional booster injection, the mice that yielded theanti-P7-1 antibody that killed 60% of IP90 spirochetes by ADCK in vitroyielded an antiserum that killed 100% of such spirochetes in anexperiment of the same kind. In addition, this same antiserum was ableto kill 50% of spirochetes from strain NT1 (a strain as yet untyped butprobably sensu stricto, as it was isolated from the cerebrospinal fluidof a patient in the North East of the United States). In contrast, nospirochetes of the JD1 strain could be killed either in vitro (by ADCK)or in vivo, in a tick-challenge experiment with mice immunized withrP7-1 and Ribi adjuvant.

[0182] ADCK was assessed both with monkey and with guinea pigcomplement. Normal guinea pig serum (from Sigma Chemical Co., St. Louis,Mo.) was used as a source of complement in the latter case. The samemonkey plasma pool from animals infected with B. burgdorferi JD1 wasused as a positive control. At a dilution of 1:10, this plasma poolkilled 57% of IP90 spirochetes after a 24 hour incubation (FIG. 3, bar1). Mouse antiserum to the rP39.5 killed 73.5% of the spirochetes at adilution of 1:10 and 60.5% at 1:50 (bars 2 and 3, respectively). Serumfrom mice immunized with Ribi adjuvant alone killed 21% of thespirochetes (bar 4). Guinea pig complement was less effective in theADCK assay. A fraction of 37% of the spirochetes was killed with thepositive control plasma at 1:10 (bar 5) whereas the mouse anti-P39.5antiserum at the same dilution killed 48% of the spirochetes in thepresence of Guinea pig complement (bar 6). A fraction of 12% of thespirochetes was killed in the presence of a 1:10 dilution of serum frommice given Ribi alone (bar 7). Interestingly, only 6% of JD1 spirocheteswere killed when incubated with a 1:10 dilution of the mouse anti-r39.5antiserum, a fraction that did not differ from that killed with justcontrol serum (not shown). Hence, the bands recognized on the JD1 blotprobably represent a spurious crossreactivity, but not the JD1 P39.5.

EXAMPLE 8 Diagnostic Use of P7-11N Humans

[0183] Nineteen human serum samples were obtained from the Centers forDisease Control and Prevention (CDC). Four of these samples were fromdonors who had no history of Lyme disease and had never resided in anarea where this disease is endemic. The other fifteen samples were frompatients who lived in Lyme disease endemic areas, had signs and/orsymptoms of the disease, and, with the exception of one patient, wereserologically positive by the criterion recommended by the CDC. Thatcriterion provides that patients be positive by a sensitive Lyme ELISAtest and be also positive by either an IgM or an IgG Western blot, suchthat at least two of the following three bands were present in positiveIgM blots (24, 39, or 41 kDa) and five of the following ten bands werepresent in positive IgG blots (18, 21, 28, 30, 39, 41, 45, 58, 66, and93 kDa).

[0184] The test, based on detection of IgG antibody by incubating serumsamples diluted at 1:200 with nitrocellulose strips onto which purifiedP39.5 antigen had been electroblotted, and subsequent detection of boundantibody by the immunoenzymatic methods described in Example 2, yieldedthe following results. The four serum samples from donors who had nohistory of Lyme disease and had never resided in an area where thisdisease is endemic had no detectable anti-P39.5 antibody. Of the fifteenremaining samples, fourteen were positive. The only negative sample wasthat which also had failed to show antibody by the criterion recommendedby the CDC. Thus, the simple procedure described herein based on theP39.5 antigen as a diagnostic probe for anti-B. burgdorferi antibody is,by this initial assessment, as sensitive and specific as the morecomplex two-step method that is currently recommended by the CDC:

[0185] In another diagnostic assay, the antigenic protein expressed fromthe 7-1 DNA fragment, rP7-1, was tested as a probe for the earlyserological diagnosis of Lyme disease in rhesus monkeys. Serum samplesobtained from three pairs of rhesus monkeys infected with threedifferent strains of B. burgdorferi, B31, JD1 and NT1, respectively,contained detectable antibody to purified rP7-1 by week 2 of infection,as detected by Western blot. Both IgM and IgG antibodies were testedsimultaneously. The result indicates that antibody to P7-1 appears earlyin the course of infection, regardless of the strain of B. burgdorferithat is eliciting the antibody response.

[0186] The sensitivity of antibody detection was assessed again byWestern blot as above, with a battery of 43 human serum samples obtainedfrom the CDC. The experiment was performed in a blinded fashion. The CDCsamples had been obtained from patients with a clinical diagnosis ofLyme disease that satisfied the stringent CDC criteria for casedefinition. Of the 43 samples from clinically positive patients, 34 werereported positive by the CDC, as judged by the Dressler criteria[Dressler, F. et al, 1993 J. Infect. Dis. 167:392-40] using the Westernblot test from MarDx Diagnostics. Inc. (Lyme disease MarBlot). With thediagnostic Western blot based on the P7-1 antigen, an equal number ofsamples were positive (34), although negative or positive results didnot always coincide in both assays. Thus, the P7-1-based Western, inwhich a single band is (or is not) detected and which is therefore atest simple to interpret, yielded the same sensitivity as one of themost accomplished, yet cumbersome to interpret, Lyme disease diagnostictests presently in the market.

[0187] In tests for antigenic specificity, so far, from 12 serum samplesof syphilitic patients, 11 were negative with the assay despite the factthat all of the samples contained antibodies that cross-reacted withmultiple antigens on a Western blot of whole B. burgdorferi antigens.The single serum sample that gave a positive result was from a patientwho also had an HIV infection and several AIDS-related infections.

EXAMPLE 9 The Cassette String Proteins

[0188] As stated above, the amino acid sequence predicted from thenucleotide sequence of the 7-1 fragment is about 50% identical to theVlsE of the B31 strain of B. burgdorferi, which is part of an antigen ofB. burgdorferi that undergoes antigenic variation by a mechanism ofrecombination whereby a central fragment of the expressed copy (VIsE) isrecombined with fragments from a string of 15 “cassettes” locatedupstream from the expressed copy [J. Zhang, cited above].

[0189] Several DNA fragments have been cloned which appear to be part ofthe cassette string of B. garinii IP90. In addition to 7-1, thesefragments, named 1-1,3-1, 6-1,9-1, and 12-1 are between 1 and 2 kb inlength. When expressed in recombinant form, substantially as describedabove for P7-1 (off of the lacZ promoter of pBluescript), each of thecassette string fragments expresses a peptide which is commensurate withthe size of the insert and which reacts with antibody from infectedmonkeys. None of the 5′ ends of these fragments contains a hydrophobicleader or a signal peptidase II consensus sequence of the type that ischaracteristic of bacterial lipoproteins. These fragments must thereforebe part of the cassette string of IP90 and, like the cassette string ofB31, they are in-frame. DNA sequences of the 5′ and 3′ termini (betweenabout 100-500 bp) of each fragment are illustrated in SEQ ID NOS: 3through 12. Note that only the 5′ end of fragment 9-1 is illustrated inSEQ ID NO: 10.

[0190] The antigenicity of these fragments has been analyzed in twoways. First, the reactivity of the fragments' Western blots with serumsamples collected longitudinally from rhesus monkeys was studied over aperiod of 48 weeks after a tick inoculation with B. burgdorferi JD1.Proteins expressed by fragments 9-1 and 7-1 reacted predominantly withserum samples collected between weeks 2 and 24 post-infection (PI)(early reactors), proteins expressed by fragments 3-1 and 6-1 reactedchiefly with serum samples collected between weeks 24 and 48 PI (latereactors), and proteins from fragments 1-1 and 12-1 showed a largelyuniform reactivity over the 48-week-long time span investigated. Thisresult suggests that each of the cloned fragments contains uniqueepitopes.

[0191] To confirm this notion, a serum pool was constructed with thesamples employed in the Western blot analysis described above, and analiquot of this “time” pool was pre-incubated with an excess of thepurified antigen expressed from 7-1 (rP7-1). In this way, the anti-P7-1antibody that was present in the serum pool was no longer available toreact with the P7-1 antigen on Western blots. Nonetheless, this serumpool was still able to react with the proteins expressed from the otherDNA fragments, with the exception of 12-1. Since the latter is a “late”reactor, it is possible that antibodies to the putatively uniqueepitopes of 12-1 may have been diluted out in the serum pool. All of theabove fragment were subcloned into the pQE vector for expression usingconventional methodologies, as described above for P7-1.

[0192] The inventor has theorized that the mechanism of antigenicvariation to which the IP90 P39.5 antigen is subjected, by virtue of itshomology to the vls E antigens of B31, indicates that this antigen iscrucially important for spirochetal survival in the vertebrate host.Thus, the methods and compositions of this invention are designed tocircumvent this spirochetal defense mechanism by immunizing a host witha large fraction of the epitopes that are expressed on the cassettestring that is the very source of the variation. This is made possibleby the unique fact that most of this cassette string is in frame.Therefore, as mentioned above, one method of vaccination involvesadministering to the host several or all of the cloned and purifiedproteins described above, which are derived from antigenicallydissimilar sections of the IP90 cassette string. See, e.g., the resultsobtained when P7-1 was introduced into a host and induced antibodieswhich killed the spirochetes.

[0193] All above-noted references and priority document are incorporatedherein by reference. Numerous modifications and variations of thepresent invention are included in the above-identified specification andare expected to be obvious to one of skill in the art. Suchmodifications and alterations to the compositions and processes of thepresent invention are believed to be encompassed in the scope of theclaims appended hereto.

1 14 1047 base pairs nucleic acid double unknown cDNA CDS 1..1047 1 AAGAAT AAT GAT CAT GAT AAT CAT AAG GGG ACT GTT AAG AAT GCT GTT 48 Lys AsnAsn Asp His Asp Asn His Lys Gly Thr Val Lys Asn Ala Val 1 5 10 15 GATATG GCA AAG GCC GCT GAG GAA GCT GCA AGT GCT GCA AGT GCT GCT 96 Asp MetAla Lys Ala Ala Glu Glu Ala Ala Ser Ala Ala Ser Ala Ala 20 25 30 ACT GGTAAT GCA GCG ATT GGG GAT GTT GTT AAG AAT AGT GGG GCA GCA 144 Thr Gly AsnAla Ala Ile Gly Asp Val Val Lys Asn Ser Gly Ala Ala 35 40 45 GCA AAA GGTGGT GAG GCG GCG AGT GTT AAT GGG ATT GCT AAG GGG ATA 192 Ala Lys Gly GlyGlu Ala Ala Ser Val Asn Gly Ile Ala Lys Gly Ile 50 55 60 AAG GGG ATT GTTGAT GCT GCT GGA AAG GCT GAT GCG AAG GAA GGG AAG 240 Lys Gly Ile Val AspAla Ala Gly Lys Ala Asp Ala Lys Glu Gly Lys 65 70 75 80 TTG GAT GCT ACTGGT GCT GAG GGT ACG ACT AAC GTG AAT GCT GGG AAG 288 Leu Asp Ala Thr GlyAla Glu Gly Thr Thr Asn Val Asn Ala Gly Lys 85 90 95 TTG TTT GTG AAG AGGGCG GCT GAT GAT GGT GGT GAT GCA GAT GAT GCT 336 Leu Phe Val Lys Arg AlaAla Asp Asp Gly Gly Asp Ala Asp Asp Ala 100 105 110 GGG AAG GCT GCT GCTGCG GTT GCT GCA AGT GCT GCT ACT GGT AAT GCA 384 Gly Lys Ala Ala Ala AlaVal Ala Ala Ser Ala Ala Thr Gly Asn Ala 115 120 125 GCG ATT GGA GAT GTTGTT AAT GGT GAT GTG GCA AAA GCA AAA GGT GGT 432 Ala Ile Gly Asp Val ValAsn Gly Asp Val Ala Lys Ala Lys Gly Gly 130 135 140 GAT GCG GCG AGT GTTAAT GGG ATT GCT AAG GGT ATA AAG GGG ATT GTT 480 Asp Ala Ala Ser Val AsnGly Ile Ala Lys Gly Ile Lys Gly Ile Val 145 150 155 160 GAT GCT GCT GAGAAG GCT GAT GCG AAG GAA GGG AAG TTG AAT GCT GCT 528 Asp Ala Ala Glu LysAla Asp Ala Lys Glu Gly Lys Leu Asn Ala Ala 165 170 175 GGT GCT GAG GGTACG ACT AAC GCG GAT GCT GGG AAG TTG TTT GTG AAG 576 Gly Ala Glu Gly ThrThr Asn Ala Asp Ala Gly Lys Leu Phe Val Lys 180 185 190 AAT GCT GGT AATGTG GGT GGT GAA GCA GGT GAT GCT GGG AAG GCT GCT 624 Asn Ala Gly Asn ValGly Gly Glu Ala Gly Asp Ala Gly Lys Ala Ala 195 200 205 GCT GCG GTT GCTGCT GTT AGT GGG GAG CAG ATA TTA AAA GCG ATT GTT 672 Ala Ala Val Ala AlaVal Ser Gly Glu Gln Ile Leu Lys Ala Ile Val 210 215 220 CAT GCT GCT AAGGAT GGT GGT GAG AAG CAG GGT AAG AAG GCT GCG GAT 720 His Ala Ala Lys AspGly Gly Glu Lys Gln Gly Lys Lys Ala Ala Asp 225 230 235 240 CGT ACA AATCCC ATT GAC GCG GCT ATT GGG GGT GCG GGT GAT AAT GAT 768 Arg Thr Asn ProIle Asp Ala Ala Ile Gly Gly Ala Gly Asp Asn Asp 245 250 255 GCT GCT GCGGCG TTT GCT ACT ATG AAG AAG GAT GAT CAG ATT GCT GCT 816 Ala Ala Ala AlaPhe Ala Thr Met Lys Lys Asp Asp Gln Ile Ala Ala 260 265 270 GCT ATG GTTCTG AGG GGA ATG GCT AAG GAT GGG CAA TTT GCT TTG AAG 864 Ala Met Val LeuArg Gly Met Ala Lys Asp Gly Gln Phe Ala Leu Lys 275 280 285 GAT GCT GCTGCT GCT CAT GAA GGG ACT GTT AAG AAT GCT GTT GAT ATA 912 Asp Ala Ala AlaAla His Glu Gly Thr Val Lys Asn Ala Val Asp Ile 290 295 300 ATA AAG GCTGCT GCG GAA GCT GCA AGT GCT GCA AGT GCT GCT ACT GGT 960 Ile Lys Ala AlaAla Glu Ala Ala Ser Ala Ala Ser Ala Ala Thr Gly 305 310 315 320 AGT GCAGCA ATT GGG GAT GTT GTT AAT GGT AAT GGA GCA ACA GCA AAA 1008 Ser Ala AlaIle Gly Asp Val Val Asn Gly Asn Gly Ala Thr Ala Lys 325 330 335 GGT GGTGAT GCG AAG AGT GTT AAT GGC ATT GCT AAG GGA 1047 Gly Gly Asp Ala Lys SerVal Asn Gly Ile Ala Lys Gly 340 345 349 amino acids amino acid linearprotein 2 Lys Asn Asn Asp His Asp Asn His Lys Gly Thr Val Lys Asn AlaVal 1 5 10 15 Asp Met Ala Lys Ala Ala Glu Glu Ala Ala Ser Ala Ala SerAla Ala 20 25 30 Thr Gly Asn Ala Ala Ile Gly Asp Val Val Lys Asn Ser GlyAla Ala 35 40 45 Ala Lys Gly Gly Glu Ala Ala Ser Val Asn Gly Ile Ala LysGly Ile 50 55 60 Lys Gly Ile Val Asp Ala Ala Gly Lys Ala Asp Ala Lys GluGly Lys 65 70 75 80 Leu Asp Ala Thr Gly Ala Glu Gly Thr Thr Asn Val AsnAla Gly Lys 85 90 95 Leu Phe Val Lys Arg Ala Ala Asp Asp Gly Gly Asp AlaAsp Asp Ala 100 105 110 Gly Lys Ala Ala Ala Ala Val Ala Ala Ser Ala AlaThr Gly Asn Ala 115 120 125 Ala Ile Gly Asp Val Val Asn Gly Asp Val AlaLys Ala Lys Gly Gly 130 135 140 Asp Ala Ala Ser Val Asn Gly Ile Ala LysGly Ile Lys Gly Ile Val 145 150 155 160 Asp Ala Ala Glu Lys Ala Asp AlaLys Glu Gly Lys Leu Asn Ala Ala 165 170 175 Gly Ala Glu Gly Thr Thr AsnAla Asp Ala Gly Lys Leu Phe Val Lys 180 185 190 Asn Ala Gly Asn Val GlyGly Glu Ala Gly Asp Ala Gly Lys Ala Ala 195 200 205 Ala Ala Val Ala AlaVal Ser Gly Glu Gln Ile Leu Lys Ala Ile Val 210 215 220 His Ala Ala LysAsp Gly Gly Glu Lys Gln Gly Lys Lys Ala Ala Asp 225 230 235 240 Arg ThrAsn Pro Ile Asp Ala Ala Ile Gly Gly Ala Gly Asp Asn Asp 245 250 255 AlaAla Ala Ala Phe Ala Thr Met Lys Lys Asp Asp Gln Ile Ala Ala 260 265 270Ala Met Val Leu Arg Gly Met Ala Lys Asp Gly Gln Phe Ala Leu Lys 275 280285 Asp Ala Ala Ala Ala His Glu Gly Thr Val Lys Asn Ala Val Asp Ile 290295 300 Ile Lys Ala Ala Ala Glu Ala Ala Ser Ala Ala Ser Ala Ala Thr Gly305 310 315 320 Ser Ala Ala Ile Gly Asp Val Val Asn Gly Asn Gly Ala ThrAla Lys 325 330 335 Gly Gly Asp Ala Lys Ser Val Asn Gly Ile Ala Lys Gly340 345 283 base pairs nucleic acid double unknown DNA (genomic) 3GCCGCTGGAT GGTGGTGAGA AGCAGGGTAA GAAGGCTGCG GATCGTACAA ATCCCATTGA 60CGCGGCTATT GGGGGTGCGG GTGATAATGA TGCTGCTGCG GCGTTTGCTA CTATGAAGA 120GGATGATCAG ATTGCTGCTG CTATGGTTCT GAGGGGAATG GCTAAGGATG GGCAATTTG 180TTTGAAGGAT GCTGCTGCTG CTCATGAAGG GACTGTTAAG AATGCTGTTG ATATAATAA 240GGCTGCTGCG GAAGCTGCAA GTGCTGCAAG TGCTGCTACT GGT 283 233 base pairsnucleic acid double unknown DNA (genomic) 4 TTTATTATAT CAACAGATTCTTAACAGTCC CTTCATGAGC AGCAGCAGCA TCCTTCAAAG 60 CAAATTGCCC ATCCTTAGCCATTCCCCTCA GAACCATAGC AGCAGCAATC TGATCATCCT 120 TCTTCATAGT AGCAAACGCCGCAGCAGCAT CATTATCACC CGCACCCCCA ATAGCCGCGT 180 CAATCGGATT TGTACGATCCGCAGCCTTCT TACCCTGCTT CTCACCACCA TCC 233 194 base pairs nucleic aciddouble unknown DNA (genomic) 5 CCGTGCAAGC TGGGTTGAAG AAGGTTGGGGATGTTGTTAA GAATAGTGAG GCAAAAGATG 60 GTGATGCGGC GAGTGTTAAT GGGATTGCTAAGGGGATAAA GGGGATTGTT GATGCTGCTG 120 AGAAGGCTGA TGCGAAGGAA GGGAAGTTGGTATGTGGCTG GTGCTGCTGG TGAAACTAAC 180 AAGGAAGCGG CCGC 194 369 base pairsnucleic acid double unknown DNA (genomic) 6 GCGGCCGCTT GAGGAAGCTGCAAGTGCTGC AAGTGCTGCT ACTGGTAATG CAGCGATTGG 60 GGATGTTGTT AAGAATAGTGGGGCAGCAGC AAAAGGTGGT GAGGCGGCGA GTGTTAATGG 120 GATTGCTAAG GGGATAAAGGGGATTGTTGA TGCTGCTGGA AAGGCTGATG CGAAGGAAGG 180 GAAGTTGGAT GCTACTGGTGCTGAGGGTAC GACTAACGTG AATGCTGGGA AGTTGTTTGT 240 GAAGAGGGCG GCTGATGATGGTGGTGATGC AGATGATGCT GGGAAGGCTG CTGCTGCGGT 300 TGCTGCAAGT GCTGCTACTGGTAATGCAGC GATTGGAGAT GTTGTTAATG GTGATGTGGC 360 AAAACAAAA 369 142 basepairs nucleic acid double unknown DNA (genomic) 7 AAGGATGGTG ATGATAAGCAGGGTAAGAAG GCTGAGGATG CTACAAATCC GATTGACGCG 60 GCTATTGGGG GTGCAGGTGCGGGTGCTAAT GCTGCTGCGG CGTTTAATAA TATGAAGAAG 120 GATGATCAGA TTGAGCGGCC GC142 210 base pairs nucleic acid double unknown DNA (genomic) 8GGTGAAACTA ACAAGGATGC TGGGAAGTTG TTTGTGAAGA AGAATGGTGA TGATGGTGGT 60GATGCAGGTG ATGCTGGGAA GGCTGCTGCT GCGGTTGCTG CTGTTAGTGG GGAGCAGATA 120TTAAAAGCGA TTGTTGATGC TGCTAAAGAT GGTGATAAGA CGGGGGTTAC TGATGTAAAG 180GATGCTACAA ATCCGATTGA CGCGGCTATT 210 236 base pairs nucleic acid doubleunknown DNA (genomic) 9 TATATAATAA AGGCTGCTGC GAAGCTGCAA GTGCTGCAAGTGCTGCTACT GGTAGTGCAG 60 CAATTGGGGA TGTTGTTAAT GGTAATGGAG CAACAGCAAAAGGTGGTGAT GCGAAGTGTT 120 AATGGGATTG CTAAGGGGAT AAAGGGGATT GTTGATGCTGCTGAGAAGGC TGATGCGAAG 180 GAAGGGAAGT TGGATGTGGC TGGTGATGCT GGTGAAACTAACAAGGAAGC GGCCGC 236 199 base pairs nucleic acid double unknown DNA(genomic) 10 ATGAGAGGAT CTCATCACCA TCACCATCAC ACGGATCCCC CGGGCTGCAGGAATTCGCGG 60 CCGCTGAAGG CTGATGCGAA GGAAGGGAAG TTGGATGTGG CTGGTGCTGCTGGTGAAACT 120 AACAAGGATG CTGGGAAGTT GTTTGTGAAG AAGAATAATG AGGGTGGTGAAGCAAATGAT 180 GCTGGGAAGG CTGCTGCTG 199 272 base pairs nucleic aciddouble unknown DNA (genomic) 11 GCCGCTGGAT GATCAGATTG CTGCTGCTATGGTTGTGAGG GGAATGGCTA AGGATGGGCA 60 GTTTGCTTTG AAGGATGATG CTGCTAAGGATGGAGATAAA ACGGGGGTTG CTGCGGATGT 120 GAAAATCCGA TTGACGCGGC TATTGGGGGTGCGGATGCTG ATGCTGCGGC GTTTAATAAG 180 GAGGGGATGA AGAAGGATGA TCAGATTGCTGCTGCTATGG TTCTGAGGGG AATGGCTAAG 240 GATGGGCAGT TTGCTTTGAC GAATAATGCT GC272 289 base pairs nucleic acid double unknown DNA (genomic) 12ACTGTTAAGA ATGCTGTTGA TATAATAAAG GCTGCTGCGG AAGCTGCAAG TGCTGCAAGT 60GCTGCTACTG GTAGTGCAGC AATTGGGGAT GTTGTTAATG GTAATGGAGC AACAGCAAAA 120GGTGGTGATG CGAAGAGTGT TAATGGGATT GCTAAGGGGA TAAAGGGGAT TGTTGATGCT 180GCTGAGAAGG CTGATGCGAA GGAAGGGAAG TTGGATGTGG CTGGTGATGC TGGTGAAACT 240AACAAGGATG CTGGGAAGTT GTTTGTGAAG AACAATGGTA ATGAGGGTA 289 142 base pairsnucleic acid double unknown cDNA CDS 2..142 mat_peptide 2 13 G CCG CTTACA AAT CCG ATT GAC GCG GCT ATT GGG GGG AGT GCG GAT 46 Pro Leu Thr AsnPro Ile Asp Ala Ala Ile Gly Gly Ser Ala Asp 1 5 10 15 CGT AAT GCT GAGGCG TTT GAT AAG ATG AAG AAG GAT GAT CAG ATT GCT 94 Arg Asn Ala Glu AlaPhe Asp Lys Met Lys Lys Asp Asp Gln Ile Ala 20 25 30 GCT GCT ATG GTT CTGAGG GGA ATG GCT AAG GAT GGG CAG TTT GCT TTG 142 Ala Ala Met Val Leu ArgGly Met Ala Lys Asp Gly Gln Phe Ala Leu 35 40 45 47 amino acids aminoacid linear protein 14 Pro Leu Thr Asn Pro Ile Asp Ala Ala Ile Gly GlySer Ala Asp Arg 1 5 10 15 Asn Ala Glu Ala Phe Asp Lys Met Lys Lys AspAsp Gln Ile Ala Ala 20 25 30 Ala Met Val Leu Arg Gly Met Ala Lys Asp GlyGln Phe Ala Leu 35 40 45

What is claimed is:
 1. An isolated, recombinant, or synthetic protein orfragment thereof that binds with antibodies to the causative agent ofLyme Disease in infected humans or animals, wherein said protein is ahomolog of a protein having the amino acid sequence formed by reading inframe the sequence of SEQ ID NO: 14 followed by SEQ ID NO: 2, andwherein said fragment comprises at least 5 consecutive amino acids inlength of said protein and wherein said protein or fragment has up tofour conservative amino acid substitutions at homologous amino acidpositions in the amino acid sequence formed by reading in frame thesequence of SEQ ID NO: 14 followed by SEQ ID NO: 2 or fragments thereof.2. The protein according to claim 1, wherein said protein is expressedby spirochetes of a B. burgdorferi sensu latostrain.
 3. The protein orfragment according to claim 1, having at least 50% identity with aprotein having the amino acid sequence formed by reading in frame thesequence of SEQ ID NO: 14 followed by SEQ ID NO:
 2. 4. The protein orfragment according to claim 1, having at least 85% identity with aprotein having the amino acid sequence formed by reading in frame thesequence of SEQ ID NO: 14 followed by SEQ ID NO:
 2. 5. The protein orfragment according to claim 1, wherein said fragment is at least eightamino acids in length.
 6. The protein or fragment according to claim 1,wherein said protein or fragment is coupled to a substrate thatimmobilizes said protein or fragment.
 7. The protein or fragmentaccording to claim 1, wherein said protein or peptide is coupled to adetectable label or signal-α-generating reagent.
 8. A kit for diagnosinginfection with a causative agent of Lyme Disease in a human or animalcomprising a protein or fragment of claim 1, and at least one of thegroup consisting of a substrate that immobilizes said protein orpeptide, a detectable label, a labeled conjugate, and a signalgenerating reagent.